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Keywords:

  • Achariaceae;
  • carbohydrate;
  • food body;
  • lipid;
  • myrmecophily;
  • plant–ant interaction;
  • Ryparosa;
  • understorey tree

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Plant food bodies are rarely observed in the field, because of continual harvesting, and are often first documented on isolated glasshouse plants. Little is known about the genus Ryparosa (Achariaceae), and the appearance of outgrowths on leaves and stems of glasshouse-raised R. kurrangii seedlings suggested that the species may produce food bodies.
  • • 
    Detailed macroimaging and histological techniques were used to characterize chemomorphological variation in food body material gathered from glasshouse plants.
  • • 
    Two distinct types of food body were observed. Multicellular pearl bodies derived from epidermal and mesophyll tissue were produced on young leaves and stems, and contained lipids and glycogen-like carbohydrates. A unique form of lipid-rich multicellular food body that ‘opens’ during development was found exclusively on mature plant tissue. A filament network was associated with food body lipid droplets.
  • • 
    This is the first detailed documentation of food body production in an understorey genus adapted to low light conditions. We suggest that the distinctive spatial deployment of Ryparosa food rewards, and the ants attracted to them, may be invaluable for keeping long-lived leaves free from epiphyllous communities.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Interactions between ants and plants that produce a more reliable (i.e. nonrandom) ant presence on plant foliage are generally mediated by one of three mechanisms – domatia, direct food resources and indirect food resources – featured alone or in various combinations (Buckley, 1982; Beattie, 1985). While ants can obtain nutrition from plants indirectly, by collecting wound sap or tending hemipteran trophobionts for honeydew, direct food rewards provided by plants are limited to extrafloral (EF) nectar and food bodies (FBs). Extrafloral nectaries are known from a considerable number of unrelated plant families, and plant–animal interactions mediated by EF nectar have been reviewed in detail (Bentley, 1976, 1977; Koptur, 1992). It has also been shown that energy-rich FBs are an important source of nutrition to ants (e.g. Janzen, 1967; Heil et al., 1997). Such structures are similarly known from a diverse range of unrelated plant taxa and can be a significant contribution to the dietary needs of ant colonies (Fiala et al., 1991; Schupp & Feener, 1991; Heil et al., 2004).

Darwin (1877) first applied the term ‘food bodies’ to the small structures found on the leaf tips of Acacia sphaerocephala and on special basal pads (trichilia) from Cecropia peltata petioles. Subsequently, Schimper (1888) suggested the use of the terms ‘Beltian bodies’ (BBs) and ‘Müllerian bodies’ (MBs) for the FBs of Acacia and Cecropia, respectively. More recently, FBs (also known as food corpuscles sensu Hölldobler & Wilson (1990), or trophosomes sensu Jolivet (1996)) have been described as small epidermal structures (Beattie, 1985; Hölldobler & Wilson, 1990), but a range of studies have concluded that they may also be composed of nonepidermal tissue (Penzig, 1893; Rickson, 1969, 1975; Buckley, 1982; O'Dowd, 1982; Heil & McKey, 2003). Many authors now use the term food body interchangeably with Beltian bodies (Heil et al., 2002, 2004), Müllerian bodies (Folgarait & Davidson, 1994, 1995; Del Val & Dirzo, 2003), and a diverse group of emergences known as ‘pearl bodies’ (PBs; Keeler, 1989; Schupp & Feener, 1991; Fiala & Maschwitz, 1992), and the current consensus seems to be that FBs can be grouped into these three subcategories.

Firstly, Beltian bodies are known only from 12 of the 13 neotropical ‘ant-acacias’ (Janzen, 1974; Seigler & Ebinger, 1995) and are produced as the final stage of leaf ontogeny at the tip of each rachis and pinnule (i.e. a modified leaflet tip; Janzen, 1967; Rickson, 1969; Buckley, 1982). The mature BB comprises differentiated living tissue with a central vascular bundle (Rickson, 1969, 1975), and once BBs have been harvested from a leaflet, they are not replaced (Raine et al., 2004). Beltian bodies contain high concentrations of protein, some lipids and a little starch (Rickson, 1969, 1975), and a recent quantification by Heil et al. (2004) confirmed carbohydrates, proteins, amino acids and lipids comprising between 15 and 25% DW BB tissue.

Secondly, Müllerian bodies are also known from a restricted range of species (Cecropiaceae), and have been thoroughly characterized in Cecropia spp. (Rickson, 1971, 1976a; Marshall & Rickson, 1973; Jolivet, 1996). Production of MBs takes place on a trichilium, which is covered in a dense mat of tannin-rich trichomes that protect the emerging growths (Rickson, 1976a, 1977). Trichilia are located on the abaxial interface of the leaf petiole and stem, and MB-producing trichilia are generally limited to the terminal two to three leaves of each branch (Longino, 1991) with an average productive life span of 3 months (Folgarait et al., 1994). Müllerian bodies are differentiated multicellular outgrowths with no vascular tissue, and are initiated in the subepidermal trichilial tissue (Rickson, 1976a,b). Müllerian bodies are unusual in that they contain a storage polysaccharide similar to glycogen, production of which is initiated when the MBs have reached 75% of full size (Rickson, 1971, 1976b; Marshall & Rickson, 1973). In addition to being rich in glycogen, mature MBs also contain significant quantities of lipids and protein (Rickson, 1976b, 1977; Belin-Depoux et al., 1997).

Lastly, pearl bodies (sensu O'Dowd, 1982) are known from a wide range of unrelated taxa (Rouppert, 1926; O'Dowd, 1982), are both morphologically and chemically heterogeneous (O'Dowd, 1980; Heil et al., 1998; Fischer et al., 2002), and are found on a variety of tissue types (Rickson, 1980; Rickson & Risch, 1984). This situation has resulted in conflicting conclusions over what constitutes a PB (Rickson & Risch, 1984; Schupp & Feener, 1991), although common features include a white lustrous colour, a spherical or elongated ‘club-like’ shape, basal constriction (i.e. a stem), and cells with a large content of lipid relative to the underlying tissue (O'Dowd, 1982). Rather than proceeding with an ever-increasing list of somewhat meaningless names (Rickson, 1980; Jolivet, 1996), O'Dowd (1982) built on the early work of Penzig (1893), Holmgren (1911) and Rouppert (1926), and proposed to group PBs into three ‘models’ according to their tissue of origin and cellular structure.

Pearl bodies have been found on almost all types of above-ground plant structures, including vegetative and reproductive tissue (Dale, 1900; Holmgren, 1911; Rickson, 1976b; O'Dowd, 1980; Fiala & Maschwitz, 1992) and on the inner walls of petiolar ant domatia (Risch et al., 1977). Pearl body tissue lacks chloroplasts (with the exception of those in epidermal guard cells, when these are present (Dale, 1901; Holmgren, 1911)) and vascular tissue (Rickson, 1980; O'Dowd, 1982) and displays all the cytological characteristics of active, living cells (Walter, 1921; O'Dowd, 1980). The chemical constituents of PBs are highly variable, not only in the type of compounds but also in their relative proportions (Penzig, 1893; Nestler, 1893; Risch et al., 1977; O'Dowd, 1980; Rickson, 1980; Rickson & Risch, 1984; Heil et al., 1998; Fischer et al., 2002).

Plant taxa providing direct food rewards span the gamut of ecosystem habitats from the deep shade of a forest understorey to high-light disturbed areas (Bentley, 1977; Buckley, 1982; O'Dowd, 1982). In tropical rainforest ecosystems, direct food rewards are more common in light gap species or species of successional communities than in shade-tolerant species (Bentley, 1976, 1977; O'Dowd, 1982; Keeler, 1989; Schupp & Feener, 1991). Thus, most understorey myrmecophytic symbioses are mediated by indirect food sources, such as hemipteran trophobionts (Stout, 1979; Fonseca, 1994; Alonso, 1998). One of the few exceptions is the Leonardoxa africana species complex (understorey trees; McKey, 2000) where domatia-dwelling Petalomyrmex ants are provided with EF nectar from large foliar nectaries (Elias, 1980; McKey, 1984, 1991, 2000; Gaume et al., 1997).

The rainforests of northern Australia are known for their low diversity of arboreal ants, relative to other tropical regions (Majer, 1990; Andersen & Majer, 2000; Majer et al., 2001). Davidson & McKey (1993) suggested that the origin of Australian rainforests may have been too recent to have allowed significant radiation of weakly competitive plant-ant genera before the arrival of other dominant ant species. Although the recent work of Blüthgen and colleagues (Blüthgen & Reifenrath, 2003; Blüthgen et al., 2004) has documented ant interactions with EF nectaries in tropical lowland rainforests of northern Queensland, to the best of our knowledge, there is no published documentation of FBs in any Australian plant.

When seedlings of the Australian rainforest endemic, Ryparosa kurrangii (Achariaceae), growing in a glasshouse developed small outgrowths on their leaves and stems, it was initially assumed they were of pathogenic origin. Foliar pesticide and fungicide sprays and plant isolation failed to prevent further outgrowths from forming, and invertebrate-induced galls could be ruled out because of the controlled conditions in which glasshouse seedlings were raised. Field observations of FBs are rare because of their continual harvesting by invertebrates (Raciborski, 1898; O'Dowd, 1979; Keeler, 1989) and FBs have been frequently discovered for the first time on tropical plants that have been raised in glasshouses (Rouppert, 1926; O'Dowd, 1980; Benson, 1985).

Food bodies are not known from Ryparosa, yet many early taxonomic papers describe the presence of ‘tubercles’, ‘verrucosities’ or ‘lenticels’ on young stems in a range of Ryparosa taxa (Sleumer, 1954). Furthermore, the documentation of stem-nesting ants in Ryparosa amplifolia (syn. R. javanica sensu Sleumer, 1954; formerly Gertrudia amplifolia (Schumann & Lauterbach, 1901; Mildbraed, 1928)), R. porcata (Jarvie & Stevens, 1998) and R. fasciculata (Moog et al., 1997; Agosti et al., 1999; Moog et al., 2003), the latter with associated pseudococcids (Heckroth et al., 1998; Agosti et al., 1999), suggests that ant–plant associations in Ryparosa may be stronger than currently thought. Although there have been no reports of associated ants in R. kurrangii, it seemed plausible that the plant growths observed in R. kurrangii may be a form of FB, and they will, hereafter, be referred to as such. The objectives of our study were to provide a detailed characterization of the developmental morphology of R. kurrangii FBs, and conduct a histological analysis of the predominant contents of FB cells. Documentation of the interactions between R. kurrangii FBs and ants, and the broader taxonomic implications of ant-plant features in Ryparosa, will be presented elsewhere (Webber et al., 2007).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Food body material

Ryparosa kurrangii B.L.Webber (Achariaceae; Flacourtiaceae pro parte) is endemic to the tropical lowland rainforests of the Daintree region in northern Queensland, Australia, and has been separated from R. javanica (Blume) Kurz ex. Koord. & Valeton sensu lato in a recent taxonomic revision (Webber & Woodrow, 2006). The subcanopy tree is an understorey specialist, maintaining leaves on branches that occur sporadically down to the base of the main trunk, regardless of light availability. New leaves are produced in distinctive synchronous flushes, concentrated around the start of the wet season in October–December (Webber, 2005).

Owing to the apparent scarcity of Ryparosa kurrangii FBs in the field, all chemomorphological characterization was performed on tissue obtained from plants raised in glasshouses. Seedlings raised from field-collected fruits sampled from multiple trees during November 2000 and November 2002 (resulting seedlings from the germination trial described in Webber & Woodrow (2004)), were planted in a standard potting mix. Approximately 60 seedlings were monitored for FB production, growing under shadecloth (10–30% of full light) in the same glasshouses and in climatic conditions similar to those outlined in Webber & Woodrow (2004). Seedlings were kept well watered and regularly supplied with liquid fertilizer (Peter's Professional Fertilizer; N : P : K 15 : 4.8 : 24.1; Scotts Australia, Castle Hill, NSW, Australia). Leaves bearing FBs were harvested and studied when the plants were between 6 and 31 months of age. At no stage were FBs purposefully removed from leaves remaining on the plants (i.e. FBs were allowed to accumulate on leaves unless they fell off).

External imaging

Morphological details of the external anatomy of FBs were examined using fresh material. Macroimages were taken with a Nikon F801 camera, fitted with an AF Micro Nikkor 105 mm 1 : 2.8 D lens. More detailed images were obtained using a Leica MZFLIII stereomicroscope fitted with a Leica DC300F digital image capture device (Leica Microsystems, Heerbrugg, Switzerland).

Internal characterization and histology

The internal characterization of FB material was performed on two distinct forms of FB identified in R. kurrangii (designated here as type A and type B FBs). Owing to the extreme fragility of type A FBs and the ease with which they detached from the plant surface, the success rate of producing prepared and stained transverse sections for light microscopy was very low. Hundreds of slides were produced to get the few images presented and similar problems have been found by other authors (Holmgren, 1911). In contrast, type B FBs were more robust and easier to section and stain. Food bodies were prepared in two ways to determine internal cellular structure and cellular contents.

Firstly, to determine cellular structure and carbohydrate contents, fresh leaf and petiole tissue bearing FBs was fixed (70% ethanol), processed on a LX120 Tissue Processor (Innovative Medical Systems Corp., Ivyland, PA, USA), and embedded in a solid matrix of paraffin and plastic polymers (Paraplast Tissue Embedding Medium, Oxford Labware, St Louis, MO, USA) using the methods of Gordon (1990). Tissue sections were cut at 8 µm on a Microm HM350 rotary microtome (Zeiss, Walldorf, Germany) and subjected to a range of staining protocols (Table 1). Potato tuber and sweet corn kernel tissue were used as controls for carbohydrate histology. Sections were air-dried and coverslipped in DPX mountant (BDH Laboratory Supplies, Poole, UK) for light microscopy and imaging.

Table 1.  Staining preparations used for fixed and fresh-frozen tissue sections of Ryparosa kurrangii food bodies and associated tissue (methods followed the references listed with any further modifications listed as footnotes)
StainTissueMethod
  • a

    Specific nuclei counterstains excluded.

  • b

    0.5% (w/v) Sudan IV in 80% ethanol, stained for 2 min.

  • c

    Counterstained with Toluidine blue for 15 s.

Toluidine blue (0.05%, pH 4.4)Fixed O’Brien & McCully (1981)
Periodic acid–SchiffFixed Cook (1990)
Best's carmineFixed Cook (1990)
Sudan black BaFresh-frozen Bayliss High (1990)
Sudan IVbFresh-frozen Ruzin (1999)
Oil red OaFresh-frozen Bayliss High (1990)
Oil red Oa+ Toluidine bluecFresh-frozen O’Brien & McCully (1981), Bayliss High (1990)
Nile blue sulphateaFresh-frozen Bayliss High (1990)
Nile blueaFresh-frozen Ruzin (1999)

Secondly, to document solvent-soluble cellular contents (i.e. lipids) in FBs, fresh-frozen sections were prepared using the methods of Bancroft (1990). Fresh leaf and petiole tissue, with FBs attached, was embedded in Tissue-Tek OCT compound (Sakura, Finetek, Torrance, CA, USA). Sections were cut at 18 µm in a Jung CM 3000 cryostat using a 2045C rotary microtome (Leica Instruments, Nussloch, Germany). This thickness was chosen as a compromise between getting high definition of cell walls and minimizing the loss of cell contents. The resulting sections were postfixed (10% neutral buffered formaldehyde; 30 s; Hopwood, 1990), air-dried and subjected to a range of staining protocols (Table 1). Fixed R. kurrangii leaf material was used as a negative control, and animal tissues were used as a positive control for lipids. Prepared sections were coverslipped in Dako Glycergel mounting medium (Dako Corp., Carpinteria, CA, USA) for light microscopy and imaging.

All light micrographs were captured on an Olympus BH2 microscope (Olympus Corporation, Tokyo, Japan) fitted with a Leica DC300F digital image capture device or a Nikon Coolscope CS1 digital microscope.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

External features

Food bodies first appeared approx. 5–6 months after seedling germination on the abaxial surface of mature leaves (Fig. 1). Leaves had generally been toughened (i.e. undergone lignification after expansion) for 1–2 wk and FBs were usually associated with the leaf midrib, nerves and tertiary venation. At this stage, seedlings were 10–20 cm tall and had 5–10 leaves. All monitored seedlings (n ∼ 60) produced FBs. As the seedlings matured and started producing leaves in distinct flushes, a large number of FBs formed on shoot tips, unexpanded leaves, mature leaves, stems and petioles. These FBs could be grouped into two distinct types based on external morphology and development.

image

Figure 1. Type B food bodies (abaxial leaf lamina) observed on young Ryparosa kurrangii seedlings raised in a glasshouse.

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Type A FBs were only found on the younger tissue of the seedlings, such as shoot tips, unexpanded leaves, expanding leaves, young leaf petioles and young green stems (Fig. 2a,b). New type A FBs formed rapidly (within 2–3 d) and were rarely initiated on tissue associated with leaves that had toughened. The bodies were pyriform to clavate in shape and up to 1.5 mm in length (Fig. 2d). Externally, type A FBs were a lustrous white colour when first formed (Fig. 2c) and became slightly yellow-brown with maturation (Fig. 2b). The surface of the bodies was irregularly dimpled (Fig. 2c,d) and there appeared to be no close association of the formation sites with leaf venation patterns or stomata. On the leaf and young stems, type A FBs were rather exposed because of the lack of pubescence (Fig. 2d). However, on the densely pubescent shoot tips and abaxial petiole swelling, type A FBs were produced between the bifurcate hairs (Fig. 2c). The point of attachment for type A FBs was narrow and the structures could be detached from the plant with minimal force (Fig. 2d). If not deliberately removed from the plant, type A FBs would shrivel up somewhat and fall off after approx. 2 wk, usually during the watering of seedlings (dislodged by the force of the water). Up to 2000 type A FBs were counted on the abaxial lamina of one fully expanded leaf, representing a density of 17.2 bodies cm−2 lower leaf surface.

image

Figure 2. Type A food bodies (FBs) observed on immature tissue of Ryparosa kurrangii seedlings raised in a glasshouse on stems (a), the leaf lamina and petioles (b). Type A FBs had a dimpled surface and were present on tissue with varying hair density (c, d), and the point of attachment was narrow (d). Bars, 500 µm (c), 200 µm (d).

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Type B FBs were distinctly different in external morphology and were observed growing only on older tissue, including brown plant stems and mature, toughened leaves. In contrast to the patterns seen in initial FB production in young seedlings (type B bodies; Fig. 1), type B FBs of larger seedlings were not specifically associated with leaf venation or stomata and were randomly scattered over the abaxial leaf surface (Fig. 3a). The point in seedling maturation at which type B FBs were first produced away from leaf veins in R. kurrangii was not determined. Type B FBs were rarely observed on the adaxial leaf surface and were only occasionally found on leaf petioles. The highest density of type B FBs was near the base of the leaf, particularly immediately adjacent to the midrib.

image

Figure 3. Type B food bodies (FBs) observed on mature tissue of Ryparosa kurrangii seedlings raised in a glasshouse. Food bodies first appeared as small bumps (a, arrows) with a smooth surface (b; u, ‘unopened’). A crack in the FB surface (b, arrow) was followed by elongation of the middle portion to form a central column with a surrounding collar (c, ‘opened’ FB). Further elongation of the column and collar produced a rather random globular mass (d). If not physically removed, FBs ended up covering the abaxial leaf surface (e) and stems (f). Bars, 1 mm.

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The growth and development of type B FBs on the abaxial leaf surface were initiated with a very small raised lump, identical in colour to the surrounding leaf tissue (Fig. 3a). As the FB matured, the lump grew in size and became golden yellow in colour (Fig. 3b). The raised lumps were globose to flattened-globose in shape and their size varied from 0.3 to 1.6 mm in diameter. At this stage, the type B FBs had a rather smooth outer surface with no noticeable dimpling. Food bodies at this stage are hereafter referred to as ‘unopened’ FBs (Fig. 3b). After approximately 1 wk, a crack appeared on the side of the lumps and the central portion started to elongate perpendicular to the leaf surface (Fig. 3b). At the same time, the base of the FB also enlarged perpendicular to the leaf surface, curling back on itself and away from the central column (Fig. 3c). Thus, when fully developed, the type B FB had a central column with a surrounding collar. Food bodies at this stage are hereafter referred to as ‘opened’ FBs (Fig. 3c). Further elongation of the column and collar produced a more random globular mass (Fig. 3d,f).

The point of attachment to underlying tissue was quite wide for type B FBs, and was usually only slightly smaller than the maximum diameter of the ‘unopened’ body. When fully expanded, type B FBs were up to 2 mm in diameter. Type B FBs were persistent on the plant unless physically removed, and were produced throughout the life span of mature (i.e. toughened) leaves, often resulting in FBs at different stages of development on the same leaf. Type B FBs were found at a considerable density on older leaves and stems (Fig. 3e,f). Some mature leaves had in excess of 1000 type B FBs on the abaxial leaf surface, representing a density of 7.1 bodies cm−2 abaxial leaf surface. Type B FBs that remained on the plant for periods in excess of 3–4 months became cream to light brown in colour (Fig. 3e,f).

Internal features

Based on fixed tissue sections, the internal structure of type A bodies was clearly multicellular (Fig. 4a). Parenchymatous tissue from the mesophyll layer was involved in type A FB production in addition to the epidermal layer (Fig. 4b). Therefore, these FBs cannot be considered as modified trichomes. It was not possible to determine the relative contribution of epidermal or mesophyll tissue cells to the final size of the mature type A FB. There was no evidence of cell wall specialization (e.g. suberization, lignification) to partition type A FB cells from lamina mesophyll cells (Fig. 4b).

image

Figure 4. Fixed tissue sections (8 µm) of food bodies (FBs) on the leaves of Ryparosa kurrangii. Foliar midrib type A FBs (a) and their point of attachment (b). Type B FBs on the abaxial leaf lamina (c; arrow, ‘open’ FB) and midrib (d). The point of attachment of type B FBs (e) and the interface between FB cells and adjacent mesophyll cells (f; arrow, abaxial epidermis). ad, adaxial leaf surface; ab, abaxial leaf surface; m, mesophyll; e, epidermal tissue; f, food body tissue. Bars, 100 µm (a); 50 µm (b, f); 250 µm (c–e).

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Type B FBs were also multicellular (Fig. 4c) and an increase in size appears to be the result of cellular division rather than cell expansion exclusively (Fig. 4d). On the outer layer of young unopened type B FBs, remains of the epidermal layer were evident (Fig. 4e). The bulk of tissue forming the central portion of the FB appears to be formed from periclinal division and radial elongation by parenchymatous cells in the underlying mesophyll layer (Fig. 4e). This cellular growth raises and eventually splits the epidermal layer during FB ‘opening’ (Fig. 4c, arrow). In mature type B FBs, there was no evidence of cell wall specialization to partition FB cells from lamina mesophyll cells (Fig. 4f).

Fresh unstained sections of leaf material with type B FBs indicated that all cells of the growths contained large, distinctive droplets and no evidence of chloroplasts. It was not possible to determine the intracellular location of these light yellow droplets. Droplets were present at all stages of growth maturity, including newly initiated and maturing FBs, and were not observed in adjacent lamina cells.

When histological stains were applied to fresh tissue sections, the nature of these droplets was revealed. Both Sudan IV and Sudan black B targeted type B FB contents, staining the distinctive droplets red and blue-black, respectively (micrographs not shown). For Sudan IV, red indicates the general presence of lipids while a blue-black reaction to Sudan black B specifically indicates nonpolar lipids (cholesterol esters or triglycerides; Bayliss High, 1990). Likewise, Oil red O stained droplets in type A FBs on petiole sections (Fig. 5a) and type B FBs on the adaxial leaf lamina (Fig. 5b). Oil red O is also known as a stain for nonpolar lipids, which appear a vivid red (Bayliss High, 1990). By counterstaining with Toluidine blue to highlight cellulosic and lignified cell walls, it was clear that droplet-containing cells were confined to the FBs and were absent from adjacent tissue (Fig. 5c). Finally, to differentiate between nonpolar lipids (e.g. triglycerides, sterol esters – pink) and polar lipids (e.g. phospholipids – blue), Nile blue sulphate was used. Stained sections confirmed that the lipids in type B FBs were nonpolar, while those in certain tissues of the leaf lamina appeared to be polar (Fig. 5d). The Nile blue methods of Ruzin (1999) produced comparable results, although with somewhat less colour intensity (micrographs not shown).

image

Figure 5. Fresh tissue sections (18 µm) of Ryparosa kurrangii food bodies (FBs) stained for the presence of lipids with Oil red O on petiolar type A FBs (a) and abaxial leaf lamina type B FBs (b); Oil red O counterstained with Toluidine blue on abaxial leaf lamina type B FBs (c) and Nile blue sulphate on abaxial leaf lamina type B FBs (d). ad, adaxial leaf surface; ab, abaxial leaf surface. Bars, 250 µm.

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image

Figure 6. Fixed tissue sections (8 µm) of Ryparosa kurrangii food bodies (FBs) stained for the presence of carbohydrates using the Periodic acid–Schiff (PAS) reaction with type A (a) and type B FBs (b), and Best's carmine for type A (c) and type B FBs (d). ad, adaxial leaf surface; ab, abaxial leaf surface. Bars, 50 µm (a, c); 200 µm (b); 100 µm (d).

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Fixed sections of leaf material with FBs were stained for the presence of carbohydrates in FB cells. Contents of type A FB cells showed a strong positive reaction to Periodic acid-Schiff reagent (staining carbohydrates a deep magenta; Cook, 1990), revealing multiple large grains per cell (Fig. 6a). In contrast, type B FBs appeared to lack significant quantities of carbohydrate (Fig. 6b), and this applied to all stages of type B FB development (micrographs not shown). Best's carmine was used to investigate the nature of the carbohydrate grains, as it is highly selective for glycogen (staining glycogen but not starch a deep red; Cook, 1990). Carbohydrate grains in type A FBs showed a strong reaction to Best's carmine (Fig. 6c), while the absence of a distinct staining response in type B FBs confirmed the previous report of a lack of carbohydrates from this tissue (Fig. 6d). Given that sweet corn, and not potato, control tissue also showed a distinct staining response to Best's carmine (micrographs not shown), there is strong evidence that the carbohydrate in type A FBs was similar to glycogen.

When cellular details of FB sections were examined at high magnification, two further features were noted. Firstly, lipid droplets of type A and B FBs stained with Oil red O appeared to be associated with a network of fine filaments (Fig. 7). The orientation of these filaments was generally parallel, well spaced and the filaments seemed to be localized in the outer region of the droplet rather than internally (Fig. 7). Secondly, FB tissue cells had distinct nuclei, indicating that the cells were most probably alive and metabolically active (Fig. 6a).

image

Figure 7. Lipid droplets and associated filament network in type A food body (FB) cells of Ryparosa kurrangii fresh-frozen tissue sections (18 µm; stained with Oil red O). Bar, 20 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Development and morphology

Based on morphology alone, it was clear that R. kurrangii produces two distinct forms of FB (type A, Fig. 2; type B, Fig. 3). These FBs were not derived from leaf tips or produced on trichilia, which ruled out any relationship to BBs or MBs, respectively. Type A FBs corresponded very closely to the definition of Model III pearl bodies (sensu O'Dowd, 1982) with a lustrous appearance, narrowed basal attachment, a club-like shape and comprising tissue of epidermal and parenchymatous mesophyll origin (Figs 2, 4). Similar structures have been described and illustrated from Hibiscus vitifolius (Dale, 1900), Cecropia peltata (Rickson, 1976a), Ochroma pyramidale (O'Dowd, 1980) and Leea hirsutus (Raciborski, 1898). In contrast, type B FBs do not conform to any previously described PB structure, in that they do not have a pronounced basal constriction, at no stage in their development are they a pearlescent colour, they are only found on mature plant tissues and they are permanent on their substrate unless physically removed (Figs 3, 4).

There is a distinct lack of studies detailing developmental morphology of FBs (for exceptions, see Rickson, 1980; Rickson & Risch, 1984), despite the fact that they have been documented in over 50 genera (Rouppert, 1926; O'Dowd, 1982). This may be, in large part, because the majority of descriptive studies were done over 100 yr ago when stylized line drawings and qualitative descriptions were standard (Meyen, 1837; Raciborski, 1898; Dale, 1900), in contrast to the microscopy techniques and structured quantification of more recent experiments. It also appears that all PBs do not conform to the general defining characteristics outlined by O'Dowd (1982), such as those of Bauhinia anatomica (Meyen, 1837) and the minute yellow ‘PBs’ of Triplaris americana that form a dusting on adaxial leaf surfaces in the absence of ants (Davidson & McKey, 1993). Thus, it may be that the definitions outlining PBs need to be broadened, or further categories of FBs might need to be defined. Alternatively, it may be that the diversity of plant FBs is too great to warrant individual names for every FB form that does not conform to the existing categories of BBs, MBs or PBs.

Cellular contents

High concentrations of nonpolar lipid droplets and carbohydrate grains provided further evidence that the structures observed on R. kurrangii were indeed FBs (Figs 5, 6). The difference between the position and morphology of type A and type B FBs was reinforced by the histological characterization of their chemical contents. Type A FBs on young tissue contained both nonpolar lipids and carbohydrates, whereas type B FBs contained considerable quantities of nonpolar lipids only. This complementary supply of FBs differing in chemical contents has also been reported in Cecropia peltata (Rickson, 1971; 1976a,b). Müllerian bodies produced on the leaf trichilia contain high concentrations of glycogen and some lipid (Rickson, 1976a,b; Belin-Depoux et al., 1997) while PBs produced on the leaves and petioles are rich in lipids with some glycogen (Rickson, 1976a). Macaranga spp. provide a similar complement of lipids and carbohydrates in a very different way. Myrmecophilic Macaranga have EF nectaries that provide EF nectar that is high in carbohydrates, in addition to lipid-rich PBs (Fiala & Maschwitz, 1991; Heil et al., 1998). However, myrmecophytic Macaranga have low carbohydrate concentrations in their EF nectar, and instead domatia-dwelling hemipteran trophobionts provide carbohydrates, in the form of honeydew, that complement lipid-rich PBs (Fiala & Maschwitz, 1991; Heil et al., 1998). The lack of EF nectaries in Ryparosa may mean that a greater range of nutritional requirements needs to be supplied by FBs to effectively maintain a significant ant presence. This is similar to the pattern seen in Piper spp. where PBs have high lipid and protein contents and no EF nectaries are present (Fischer et al., 2002). Notably, hemipteran tending by the plant-ant Cladomyrma has been documented in R. fasciculata, thereby providing an alternative source of carbohydrates in the absence of EF nectaries (Heckroth et al., 1998; Agosti et al., 1999).

The composition of R. kurrangii FB contents and the contents of associated cells are notable for two further reasons. Firstly, the occurrence of carbohydrates in type A FBs showing a positive reaction to Best's carmine (a glycogen-selective stain; Cook, 1990) is unusual in plants (Fig. 6). Glycogen-like storage carbohydrates in plants have previously only been described from Zea mays seeds and Cecropia MBs (Peat et al., 1956; Archibald et al., 1961; Rickson, 1971; Marshall & Rickson, 1973; Belin-Depoux et al., 1997), as starch is usually the common form of plant carbohydrate storage. Further chemical characterization of R. kurrangii FB carbohydrates is now underway to confirm these histological results. Secondly, the association of FB lipid droplets with an arrangement of fine filaments (Fig. 7) appears to be similar to that of lipid droplets described from the Beltian bodies of Acacia spp. (Rickson, 1975). Rickson (1975) highlighted the difference between the Acacia filaments and the protein–lipid boundary layer that usually surrounds plant lipid droplets (e.g. oleosins, caleosins; Huang, 1992; Murphy, 1993; Weselake, 2005). A similar complex of fine, highly ordered filaments (vimentin intermediate filaments, sensu Franke et al., 1987) has been observed in adipose cells of animal tissue at the lipid droplet–cytoplasm interface (Wood, 1967; Franke et al., 1987; Almahbobi, 1995; Lieber & Evans, 1996). These filaments were originally hypothesized to offer a form of support for the large lipid droplets, as there was no specific membrane separating the lipid droplet from the rest of the cytoplasm (Wood, 1967; Rickson, 1975). However, more recent work conducted on adipose tissue suggests the primary importance of vimentin intermediate filaments in the formation and enlargement of lipid droplets (Franke et al., 1987; Lieber & Evans, 1996). Notably, FB lipids and their associated filaments have not been mentioned in more recent reviews that cover lipid body formation in higher plants (Murphy & Vance, 1999; Zweytick et al., 2000; Murphy, 2001, 2005).

Food body production in the understorey

This characterization of FB production by R. kurrangii appears to be the first confirmed report of FBs in a taxon that is largely restricted to the understorey of primary forests. Within Ryparosa, there is considerable variation in the intimacy and trophic structure of ant–plant relationships from myrmecophilic to myrmecophytic; however, all species produce food bodies (Webber et al., 2007). In their substantial review of myrmecophytic ant plants, Davidson & McKey (1993) list 59 myrmecophytic plant genera that are predominantly associated with ‘primary forest’. In their substantial review of myrmecophytic ant plants, Davidson & McKey (1993) list 59 myrmecophytic plant genera that are associated with ‘primary forest’. Of these species, 58% involved hemipteran trophobionts, 22% produced EF nectar and 8% (five genera) produced FBs (32% had no known food rewards and some plants had more than one food type). However, of the six primary forest genera that are documented as producing FBs (the authors did not list Ryparosa as having FBs), Macaranga, Piper, Pourouma and Cecropia are known to be more commonly found in canopy gaps or disturbed areas (Risch et al., 1977; Fiala et al., 1989; Yu & Davidson, 1997; van der Meer et al., 1998; Fonseca, 1999; Del Val & Dirzo, 2003). Moreover, Pourouma is known to depend on high light to complete its life cycle (Rijkers et al., 2000). The only other primary forest genus claimed by Davidson & McKey (1993) to produce FBs is Cordia, and FBs have only been reported from one species, the understorey treelet Cordia nodosa (Solano et al., 2005). Solano et al. (2005) present preliminary evidence that these structures, while functioning as EF nectaries, may also share functional similarities with FBs. However, applying the term ‘food body’ to these structures may be somewhat premature, given that they also appear to share many similarities with ‘Hochnektarien’ EF nectaries (sensu Zimmermann, 1932). Thus, there appears to be a significant paucity of FB-producing plant genera adapted to a continual existence in the low-light conditions of the rainforest understorey.

Food body production in R. kurrangii is known to be associated with an increased presence of opportunistic ants, which display patrolling and territorial behaviour while harvesting FBs (Webber et al., 2007). The production of type A FBs on young foliage supports the theories of resource allocation (optimal defence theory; McKey, 1974, 1979; Rhoades, 1979), which hypothesize that plant resources will be differentially allocated towards defending more vulnerable and valuable plant parts (i.e. young leaves). However, the continual production of type B FBs on toughened mature leaves and older stems appears to be unique to Ryparosa, and at odds with the theories applied to ant-mediated defence (McKey, 1984, 2000). So why would a rainforest plant invest considerable resources in producing FBs on older leaves? Ryparosa is an understorey tree, growing primarily in low-light conditions, where the importance of maintaining optimum photosynthetic functionality of older foliage is critical. Thus, the defence strategies deployed by this taxon should reflect the ecological pressures of the understorey environment (Fonseca, 1994).

Effective ant defence, such as ‘leaf-cleaning’ (McKey, 1984) or acting as ‘bodyguards’ against animal herbivores (Fiala et al., 1989), requires continual provision of ant-attracting mechanisms by the host plant. In plants that lack domatia, this translates to a reliable source of food rewards. For pioneer species in gap conditions, fast growth rates and a relatively continual production of young leaves ensure that the associated ant rewards (EF nectar, FBs) are consistently present (Coley & Kursar, 1996). Yet in the rainforest understorey, the majority of plants, including Ryparosa, produce new leaves in synchronous bursts (flushing; Reich, 1995; Kursar & Coley, 2003). However, if ant food rewards are also produced continually on older foliage, the likelihood of consistent ant patrolling on an understorey plant, with infrequent new leaf production, is increased. Moreover, the costs relative to the benefits of ant-mediated leaf cleaning defence in an understorey environment may be justified in plant taxa with long leaf life spans. In these low-light conditions, an ant presence on plants may confer protection to the plant primarily as leaf cleaners, and their role as bodyguards may be secondary, especially in nonmyrmecophytic species.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Doug Yu and Doyle McKey for intellectual support, and Dennis O'Dowd, Martin Heil and two anonymous reviewers for providing valuable advice on earlier versions of this manuscript. BLW was a recipient of an Australian Postgraduate Award PhD Scholarship. Field sampling was conducted under Scientific Purposes permits issued by the Queensland Department of Environment and Heritage (F1/000233/00/SAA, WISP01144103, WITK01149403) and with the full permission and support of private landholders when collected on freehold land.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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