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Keywords:

  • ferralitic oxisol;
  • hypermagnesian;
  • Mn hyperaccumulator;
  • photosynthetic layer;
  • qualitative scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) mapping

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Hyperaccumulation by plants is a rare phenomenon that has potential practical benefits. The majority of manganese (Mn) hyperaccumulators discovered to date occur in New Caledonia, and little is known about their ecophysiology. This study reports on natural populations of one such species, the endemic shrub Maytenus founieri.
  • • 
    Mean foliar Mn concentrations of two populations growing on ultramafic substrates with varying soil pHs were obtained. Leaf anatomies were examined by light microscopy, while the spatial distributions of foliar Mn in both populations were examined by qualitative scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS).
  • • 
    Plants growing on two different substrates were found to have very different mean dry weight (DW) foliar Mn concentrations. Light microscopy showed that the leaves had very distinct thick dermal structures, consisting of multiple layers of large cells in the hypodermis. In vivo X-ray microprobe analyses revealed that, in both populations, Mn sequestration occurred primarily in these dermal tissues.
  • • 
    The finding here that foliar Mn is most highly localized in the nonphotosynthetic tissues of M. founieri contrasts with results from similar studies on other woody species that accumulate high Mn concentrations in their shoots.

Introduction

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

Hyperaccumulators are plants with an extreme tolerance to specific heavy metals and metalloids that accumulate in their aerial parts in concentrations greatly exceeding ‘normal’ thresholds of toxicity (Brooks et al., 1979; Baker, 1981; Baker & Brooks, 1989). The soils of their native habitats generally have elevated concentrations of these elements, and include the type termed ultramafic or serpentine. Hyperaccumulation is a rare phenomenon amongst higher plants. It is of inherent interest, and is seen as exploitable, particularly for the purposes of phytoremediation and phytomining (Baker et al., 2000; Brooks & Robinson, 2000). Over 450 hyperaccumulator taxa are known, collectively comprising < 0.2% of angiosperms. Of these, nearly three-quarters hyperaccumulate Ni (Baker & Brooks, 1989), which are the most widely researched. Hyperaccumulators of other metals and metalloids have been, to date, comparatively less well investigated, while some remain altogether unstudied.

Manganese hyperaccumulation has been arbitrarily defined by a threshold foliar concentration of 10 000 µg g−1 dry weight (Baker & Brooks, 1989). Plants that hyperccumulate Mn are predominantly woody and hence unsuited to short-term controlled study. The number of species has varied with taxonomic changes, and currently nine are recognized worldwide (Reeves & Baker, 2000; Bidwell et al., 2002; Xue et al., 2004). They include seven endemics from New Caledonia. The existence of the New Caledonian Mn hyperaccumulators was noted in the literature almost three decades ago with little follow-up study (Jaffré, 1977, 1979). More recently, Gossia bidwillii (Benth.) N. Snow & Guymer (Myrtaceae) from Australia and Phytolacca acinosa Roxb. (Phytolaccaceae) from China were found to be Mn-hyperaccumulating species (Bidwell et al., 2002; Xue et al., 2004). The heterogeneity of the Mn-hyperaccumulative trait was studied in P. acinosa, a herbaceous species, under controlled conditions (Xue et al., 2005), and in natural populations of the tree G. bidwillii (Fernando et al., 2007). The trait was found to be constitutive in the former, while the latter found Mn hyperaccumulation to be highly heterogeneous in G. bidwillii growing in its native habitats on a variety of substrates, including serpentine. Microprobe studies have demonstrated that in G. bidwillii, primary localization of foliar Mn is in the photosynthetic tissues (Fernando et al., 2006a,b). This contrasts markedly with findings on other hyperaccumulating plants, including P. acinosa, where the highest localized concentrations of foliar Ni, Zn, Cd, As and Mn have been found to be in the nonphotosynthetic tissues (Vázquez et al., 1992; Küpper et al., 2000; Mesjasz-Przybylowicz et al., 2001; Lombi et al., 2002; Robinson et al., 2003; Bidwell et al., 2004; Xu et al., 2006). The more recently discovered Mn hyperaccumulators renewed interest in the New Caledonian species, leading to Mn localization studies on Virotia neurophylla (Guillaumin) Virot (Proteaceae), an endemic Mn-hyperaccumuating species. Like G. bidwillii, it was found to have an unusual spatial distribution of foliar Mn (Fernando et al., 2006b), where primary sequestration occurs in the palisade mesophyll cells.

The flora and biodiversity of New Caledonia are characterized by a very high degree of endemism, largely because of its metalliferous serpentine soils (Jaffré, 1980; Proctor, 2003), and includes the Mn hyperaccumulator Maytenus founieri (Panch. & Sebert) Loesn. (Celastraceae). Jaffré (1977) observed that several Maytenus species in the field hyperaccumulated Mn independently of soil pH. In that early study, the taxonomy of Maytenus included at least four species; however, all New Caledonian Maytenus species are currently grouped under founieri. This includes the two taxa, M. founieri ssp. founieri (Pancher & Sebert) Loesn. and M founieri ssp. drakeana (Loesn.) I.H. Mueller ((Pancher & Sebert) Loesn., Fl. N. Caled., 20:73 (1996)).

Manganese is an essential plant micronutrient, which is taken up in the +2 oxidation state (Clarkson, 1988). It is often present in soil in the form of insoluble higher oxides of the +4 and +3 valency states (Gilkes & McKenzie, 1988). These can lead to the +2 form not only by acidification of the soil solution but also by extreme climatic conditions which give rise to soil waterlogging, heating and drying (Grasmanis & Leeper, 1966; Siman et al., 1974; Heenan & Carter, 1977), as well as by the activities of anaerobic and aerobic microorganisms. Plants growing in basic soils can take up Mn by mycorrhizal or root exudate reduction in the rhizosphere (Leeper & Uren, 1997).

The observation by Jaffré (1977) that Maytenus hyperaccumulated Mn independently of soil pH indicates there may be a specific mechanism employed by M. founieri, which facilitates this. There have been no further studies to verify this, for example, by testing populations of M. founieri growing on substrates with differing acidities. This present investigation sets out to view the Mn-hyperaccumulative trait in two populations of M. founieri growing on two substrates differing in Mn content as well as pH. Using X-ray microanalysis to generate qualitative elemental maps, this study examines for the first time the spatial distribution of foliar Mn in M. founieri. Localization studies were carried out on both populations to assess the species-wide variability of this sequestration pattern.

Materials and Methods

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

Maytenus founieri is a compact shrub, approx. 0.6–3 m tall, with sclerophyllous obovate leaves, and inflorescences of small waxy, cream flowers. It is widely distributed on Grande Terre, the main island of New Caledonia. Sampling for the purposes of this study was carried out in the south of the island, around Nouméa. Two Maytenus groves were selected on the basis of their substrates. These plants appeared healthy and displayed no signs of physiological stress.

The ultramafic host substrates may be described as follows: site 1, brown eutrophic hypermagnesian soil (vertic cambisol) on serpentinite; site 2, ultramafic oxisol (ferrallitic soil of the ferritic type) on peridotite (Proctor, 2003). Properties such as pH, Mg concentrations and physical appearance have long been recognized as consistent distinguishing features of these two distinct soil types (Jaffré & Latham, 1974; Jaffré, 1976, 1980). Their pedological evolution can be traced back to extremities on a geological timescale. The soil on site 1 was reddish brown, shallow and stony with irregular sizes. Site 2 had dark blackish-brown shallow gravel made up of relatively uniformly sized rounded stones. Coordinates for sites 1 and 2 were 22°17′32″S, 166°39′10″E and 22°18′71″S, 166°45′57″E, respectively. The habitat of site 1 is known as ‘maquis arbustif’, and occurs at the base of mountains. It comprises thicket of varying density, with small shrubs and low understorey. Maytenus was found interspersed with Tarenna microcarpa (Rubiaceae), Stenocarpus milnei (Proteaceae), Xanthostemon multiflorus (Myrtaceae), Soulamea pancheri (Simaroubaceae), Hibbertia vieillardii (Dilleniaceae). Site 2 is known as ‘maquis buissonant’, and has a more or less continuous cover of much-branched shrubs, with virtually no understorey herbs. The associated species were Tristaniopis callobuxus, T. guillainii (Myrtaceae), Gymnostoma deplancheanum (Casuarinaceae) and Alstonia lenormandii (Apocynaceae). Grevillea gillivrayi (Proteaceae), Longetia buxoides (Euphorbiaceae) and Alphitonia neocaledonica (Rhamnaceae) were found on both sites. M. founieri ssp. founieri occurred on site 1, and M. founieri ssp. drakeana on site 2.

Each site was sampled on two separate occasions: on the first, branchlets were collected from 10 shrubs, and five soil samples were taken from the top 10 cm layer. This was for analysis of plant and soil samples, and to carry out soil pH tests. On the second visit, leaves were sampled into humid packaging (zip-lock plastic bags containing damp paper towels) from three shrubs at each site so as to preserve field freshness required for prompt subsampling in the laboratory before cryofixation for Mn localization studies.

Inductively coupled plasma analyses and soil pH measurements

Leaf samples were washed and oven-dried at 70°C for ICP-OES (inductively coupled plasma optical emission spectroscopy) analysis. Owing to the small leaf size and density of foliation, branchlets were stripped of all fully expanded leaves, which were washed, dried, ground and sampled to approx. 0.3 g. For each plant, fully expanded leaves were pooled to include young and mature leaves, and digested in 5 ml 70% nitric acid at 125°C for 2 h. Digests were treated with 30% hydrogen peroxide, diluted to 75 ml with distilled water, analysed and quantified against a series of aqueous standards acidified with nitric acid. Soil samples were oven-dried at 70°C for ICP analyses (total and extractable Mn content). For total concentrations, approx. 0.2 g soil was digested in 5 ml of aqua regia (a 1 : 3 mixture of 70% HNO3 and 42% HCl) at 130°C for 3 h. They were then processed in the way that the leaf digests were, using standards acidified with aqua regia for quantification. Using this methodology, dry weight (DW) concentrations of Mn, Mg, and Ca in plant and soil samples were obtained. These elements were analysed because Mn was of major interest in this study, and Mg : Ca ratios in serpentine substrates are generally known to be elevated. To assess soil extractable Mn, 5 g dry soil and 25 ml 0.05 m EDTA (pH 6.0) were shaken for 4 h, and the filtered supernatant analysed by ICP-OES. Soil pH was measured after 5 g of dried soil was shaken with 25 ml of distilled water for 2 h. Statistical analyses were carried out on resulting data, using SPSS 12.0.1 for Windows (SPSS Inc., Chicago, IL, USA). One-way analyses of variance (anova) were applied to the leaf, soil and pH data.

Light microscopy

The mid-transverse areas of fully expanded fresh leaves were hand-sectioned directly into 50% ethanol, and later placed in a droplet between a glass slide and cover slip for examination by light microscopy.

Qualitative SEM/EDS elemental mapping

The mid-transverse areas of fully expanded fresh leaves of comparable maturity were sampled from each of three shrubs on sites 1 and 2. Leaf fragments (approx. 1 × 0.5 cm) were excised and rapidly frozen in melting nitrogen (CT1501, Oxford Instruments, Eynsham, UK). They were then freeze-dried. Dried tissues were hand-sectioned with previously unused and cleaned razor blades to expose cross-sectional surfaces. Thick slices (sections) were attached to aluminium sample mounts with conductive carbon tape, platinum-coated (∼2 nm), and stored desiccated before analysis. At least five sections per sample were mounted so as to enable selection of a single area for X-ray mapping, on the basis of obtaining an SEM image with minimal charging.

Mapping was carried out on a JEOL JSM 840 A scanning electron microscope (JEOL, Tokyo, Japan), fitted with a Link exL X-ray analyser and LZ5 detector (Oxford Instruments, Wycombe, UK). Secondary electron images and qualitative maps were obtained at 15 kV, with a beam current of 5 × 10−9 A. Qualitative maps of Mn, K, and Mg and their background maps were collected simultaneously, using peak energy windows and corresponding continuum windows set on the low-energy side of the peaks.

Results

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

As shown in Table 1, the mean foliar Mn and Ca concentrations at site 1 were significantly lower than at site 2 (P < 0.0001), while the reverse was observed for Mg (P < 0.0001). Each of the 20 individual leaf samples used to calculate means in Table 1 was obtained from a sample pool of fully expanded young to mature leaves; hence the aforementioned significant differences in foliar concentrations are not an age effect resulting from inconsistent sampling practice. Mean soil pH, total Mn, Mg, Ca and extractable Mn concentrations for soils on sites 1 and 2 are also recorded in Table 1. These data demonstrate that site 2 has the more acidic substrate (P < 0.0001), and that total Mn (P = 0.015), extractable Mn (P = 0.002) and Mg (P = 0.044) concentrations are higher in the surface soil horizon of site 1 than that of site 2. No significant difference in soil Ca concentrations was observed between sites (P = 0.079). The large standard error values attached to these mean concentrations are evidence of the heterogeneity of the substrates on both sites. The mean total soil Mn concentration of site 2 includes a high outlier of 2373 µg g−1. The results of soil analyses obtained here are in agreement with the data of Jaffré (1976, 1980), who recorded mean pH values of 6.75 (n = 19) and 4.91 (n = 18) for the two soil types labelled 1 and 2 in this study. Those soils were sampled from locations other than sites 1 and 2 of the present study. The defining soil characteristics of pH, Mg concentration and colour in the present study are consistent with existing knowledge on these soil types.

Table 1.  Mean leaf and soil concentrations, and soil pH
SiteSampleMn (tot.)Mn (ex.)Mg (tot.)Ca (tot.)pH
  1. Mean values (± SE) (µg g−1 DW); leaf, n = 10; soil, n = 5.

  2. tot., total; ex., extractable.

1Leaf6650 ± 716  17 130 ± 171013 110 ± 799
Soil6960 ± 19203010 ± 6601 000 670 ± 42 0602770 ± 10906.37 ± 0.16
2Leaf20 060 ± 2715110 ± 40929 330 ± 1860
Soil944 ± 36333 ± 17288 ± 59563 ± 1314.46 ± 0.11

Light microscopy

Light microscopy (Fig. 1) shows that the mesophyll contains multiple palisade cell layers, and is capped by an extremely thick, nonphotosynthetic dermal layer, which occupies approx. 20–30% of the total leaf thickness. This dermal structure consists of multiple layers and has been referred to as multiseriate (Fritch & Salisbury, 1920; Fahn, 1974). The top layer resembles a ‘normal’ epidermal cell layer beneath the cuticle, while the hypodermis consists of two rows of large cells.

image

Figure 1. Light micrographs of Maytenus founieri leaf cross-sections taken from site 1(a–c) and site 2 (d–f). Note the thick upper epidermis (ue), and multiple palisade mesophyll cell layers (p) in all samples.

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Qualitative SEM/EDS maps

Cross-sections of one leaf from each of three different shrubs per site are shown in Figs 2 and 3. The maps obtained for all six specimens had similar qualitative spatial distributions of Mn, Mg and K, with Mn most highly localized in the dermal structures of both subspecies. Note that these qualitative data were obtained from leaf samples of comparable maturity. There is some difference in Mn distribution throughout the leaf mesophyll between sites 1 and 2. Manganese was detected in the mesophyll of plants sampled from site 1, and not in the mesophyll of site 2 samples. This observation was consistent in the replicates examined, and is represented in Fig. 4, which shows line profile plots of brightness intensity corresponding to the white bars across the Mn maps in Figs 2 and 3. These profiles are directly proportional to the intensity Mn X-ray counts.

image

Figure 2. Scanning electron microscopy (SEM) image and corresponding Mn, Mg and K X-ray maps of a site 1 Maytenus founieri leaf cross-sectional surface (upper epidermis above). Asterisks indicate a lower margin of cross-section. The white bar gives the position of the line profile in Fig. 4.

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image

Figure 3. Scanning electron microscopy (SEM) image and corresponding Mn, Mg and K X-ray maps of a site 2 Maytenus founieri leaf cross-sectional surface (upper epidermis above). The white bar gives the position of the line profile in Fig. 4.

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image

Figure 4. Line profiles (a) and (b) correspond to white bars across Mn energy dispersive spectroscopy (EDS) maps in Figs 2 and 3, respectively (upper epidermis LHS). They indicate the brightness intensity of the maps, which is proportional to Mn X-ray counts.

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Magnesium was detected in situ in all leaf cross-sections (Figs 2, 3), and found to be most concentrated in the apoplast of their dermal tissues. Comparison of Mn and Mg distribution through these upper dermal structures showed that the former was more evenly spread in comparison to corresponding Mg maps, in which cell shapes were negatively outlined. The K maps have been included to demonstrate retention of sample integrity, at least to the cellular level.

Discussion

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

As can be seen from Table 1, the results obtained in this study show that DW foliar Mn concentrations of M. founieri on sites 1 and 2 differ considerably. According to these data, M. founieri is strongly Mn-hyperaccumulating on the acidic ferralitic oxisol of site 2, but not on the hypermagnesian substrate of site 1. This is despite the lower Mn concentration of site 2 soil, and indicates the ability of M. founieri to accumulate bioavailable Mn in its foliage. The present findings for M. founieri differ markedly from those of Jaffré, 1977), who concluded that foliar Mn accumulation by New Caledonian Maytenus was determined only by the soil Mn concentration and was independent of soil pH. This discrepancy may be caused by one or more of the following: (i) taxonomic changes in Maytenus may be masking functional differences between subspecies; (ii) samples for the two studies were not taken from the same sites; (iii) there may be variable effects on soil Mn availability because of factors such as humus content, soil hydromorphy and microbial activity in the rhizosphere.

The discrepancy between the outcomes of the previous and present studies could be resolved by further sampling plants and soils from more sites, and including different climatic conditions for each site. Here, the observed variation in foliar Mn concentrations between the two ecotypes of M. founieri might be attributed to genetically determined differences, as found in some of the herbaceous hyperaccumulators (Baker et al., 1994; Macnair, 2002; Pollard et al., 2002), and more recently in the Mn-hyperaccumulating tree G. bidwillii (Fernando et al., 2007). The heterogeneous nature of the soil is reflected in the high standard error values obtained (Table 1). Furthermore, soils sampled from the top 10 cm layer are unlikely to share exactly the same characteristics of those surrounding the roots of M. founieri. Although entire plant populations were not sampled on sites 1 and 2, it is noteworthy that the variation of Mn hyperaccumulation within each leaf sample group was not large, and is reflected in the small standard error values of Table 1. In contrast, population sampling for the investigation of Mn hyperaccumulation in G. bidwillii demonstrated the trait to be highly heterogeneous (Fernando et al., 2007).

Recent in situ microprobe studies on four tree species showed that the highest localized foliar Mn concentrations occur in their photosynthetic tissues (Fernando et al., 2006a,b), whereas similar studies on herbaceous hyperaccumulators have found that nonphotosynthetic tissues contain the highest localized concentrations of Ni, Zn, Cd, As and Mn (Vázquez et al., 1992; Küpper et al., 2000; Mesjasz-Przybylowicz et al., 2001; Lombi et al., 2002; Robinson et al., 2003; Bidwell et al., 2004; Xu et al., 2006). Gossia bidwillii and Virotia neurophylla are Mn hyperaccumulators, while Macadamia integrifolia and M. tetraphylla accumulate high foliar Mn concentrations below the threshold value for Mn hyperaccumulation. The leaf anatomies of all four species consist of multiple palisade layers, where Mn has been found to be sequestered in the highest concentrations. It was argued that since palisade cells are highly vacuolated and are present in multiple layers, their combined vacuolar volume is substantial enough to provide sequestration sites for excess Mn (Fernando et al., 2006b). Two decades before these studies, foliar Mn in the Mn-accumulating species Eleutherococcus sciadophylloides and Ilex crenata from Japan was investigated in situ, and it was demonstrated that primary sequestration was in the nonphotosynthetic tissues, with some distribution through the multiple palisade-layer mesophyll (Memon & Yatazawa, 1980; Memon et al., 1981). Of the existing localization studies on the foliar distributions of other hyperaccumulated metals/metalloids, there is little description of overall leaf anatomies. Where this information has been made available, showing a multiple-palisade layer leaf anatomy, primary sequestration is in the nonphotosythetic dermal tissues (Broadhurst et al., 2004; McNear et al., 2005). Multiple palisade-layer leaf anatomy is determined by genetics and growth conditions of drought and/or high sunlight. Given the major role of Mn in the light reactions of photosynthesis, and that palisade cells are photosynthetically active, it has been suggested (Fernando et al., 2006a,b) that there may be an association between a multiple palisade-layer leaf anatomy and Mn accumulation in the mesophyll.

In this study, the leaf anatomy of M. founieri was examined by light microscopy and found to be very distinctive, particularly by its extremely thick dermal structure consisting of a double-layer hypodermis, made up of large cells. This feature is thought to be a xerophytic plant adaptation, which allows for the storage of water (Fritch & Salisbury, 1920; Fahn, 1974). Qualitative SEM/EDS mapping showed (Figs 2, 3) that foliar Mn was mostly concentrated in this structure. Although ultrastructural studies are required to resolve subcellular detail, it is reasonable to hypothesize that in M. founieri leaves, these large dermal cells have large vacuoles where excess Mn can be sequestered. Here, there is no apparent link between excess Mn sequestration and photosynthesis, or light harvesting. It is possible that the palisade cell layers, which occupy 30–50% of the leaf thickness, may provide less collective vacuolar volume, and that this is a stronger determinant in the overall strategy of foliar detoxification of excess Mn. There is no clear explanation for the observed difference in Mn distribution through the leaf mesophyll of site 1 and 2 plants (Figs 2–4). It may be related to the disparity in their overall foliar Mn concentrations, that is, site 1 plants had a significantly lower mean leaf Mn concentration and were found to have more Mn in their mesophyll tissue. The hypothesis that the spatial distribution of Mn in the leaves of M. founieri is, to some degree, governed by their leaf Mn concentrations warrants further investigation.

Several mechanisms for foliar detoxification of metals have been suggested, most commonly sequestration in nonphotosynthetic dermal tissue, and often in the vacuoles or leaf hairs/trichomes of these dermal layers (Krämer et al., 1997; Gonzalez & Lynch, 1999; Küpper et al., 2000; Robinson et al., 2003; Broadhurst et al., 2004). Excess foliar Mn is known to be sequestered in the leaf hairs of certain tolerant crop plants, and the occurrence of simultaneously highly localized Mn and Si in leaf trichomes has been interpreted as possible evidence of the amelioratory effects of Si (Horst & Marschner, 1978; Blamey et al., 1986; Horiguchi, 1988). Guttation has also been suggested as a possible mechanism of Ni disposal in the hyperaccumulator Alyssum murale (McNear et al., 2005). It is feasible that more than one mechanism may be available to a plant, and hence factors such as leaf anatomy, toxicity of the accumulated element, growth conditions, seasonal variations and special adaptations may have a combined effect in determining foliar detoxification strategies.

In conclusion, M. founieri was found to exhibit intraspecific variation in its accumulation of high foliar Mn concentrations on acidic and basic soils. Strong Mn hyperaccumulation only occurred on the acidic substrate, and this could be the result of differences in ecotype and/or substrate. Manganese was located in situ in the leaves, where it was found to be most highly concentrated in the dermal tissues of both populations examined. The suitability of M. founieri for long-term remediation of high-Mn acidic and basic soils needs to be assessed by propagation and Mn treatment trials.

Acknowledgements

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

The authors are extremely grateful to Alexandre Lagrange (IRD, Nouméa) for his botanical expertise in the field. The help of our late colleague Nicolas Perrier (IRD, Nouméa) was also invaluable. All work carried out in this manuscript was in compliance with current Australian laws.

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