DISCUSSION
This study details the first-ever growth experiment on the Mn hyperaccumulating tree, G. bidwillii , exposed here to a range of Mn treatments and reports for new discovery of the Mn hyperaccumulative trait in wild G. acmenoides , on the highly acidic Mn-rich soils is intriguing given past analyses of samples sourced from a range of other eastern Australian locations had not detected Mn hyperaccumulation in this species (Fernando et al. 2009b). Foliar Mn concentrations in experimental G. bidwillii to as high as ~3-fold Mn hyperaccumulation threshold of 10 000 µg g-1 (van der Ent et al. 2013) aligns with field data from earlier studies of this species (Bidwell et al. 2002; Fernando et al. 2006b). A weak relationship between substrate Mn supply and foliar Mn concentrations, most notably, high Mn uptake by control plants is also consistent with past field observations (Bidwell et al., 2002, Fernando et al., 2006b, Fernando et al., 2007). The ability of Gossia bidwillii as shown experimentally here and as previously noted in the field to vastly over-accumulate Mn even in very low soil-supply is a common characteristic hyperaccumulation, i.e., the ability to scavenge from host substrates (Baker 1981; (Fernando et al., 2007).
Contrasting findings of previous field studies that have not foundG. acmenoides to hyperaccumulate Mn, and this present field study describing strong Mn hyperaccumulation by this species may be attributable to genetic differences, as also observed in Denhamia founieri from New Caledonia by Fernando et al. (2008). The present findings on G. acmenoides suggests heterogeneity across the species, warranting further investigation of the genetic basis of Mn hyperaccumulation in Gossia, for example, as interrogated by Pollard et al (2002) for ubiquitous metal hyperaccumulating herbs of the Northern Hemisphere. Variability of Mn hyperaccumulation trait inG. bidwillii has previously been reported, however there is greater consistency across its broad natural range in comparison to the emerging picture of G. acmenoides (Fernando et al., 2007). Heterogeneity of metal accumulation has also been described in several species of hyperaccumulators of metals other than Mn (Pollard et al., 2002, Baker et al., 1994, Macnair, 2002). The extreme acidity of the host soil (pH 4.20, Table 3) from which G. acmenoides was sampled for this study warrants consideration in the context of Mn availability at the root-soil interface. It is plausible that the apparently high variation in Mn accumulation by G. acmenoides reflects specific rhizosphere effects such as acidification and/or microbial associations unique to a particular site location that renders soil-Mn highly bioavailable. These findings yet again highlight the gaps in knowledge around metal hyperaccumulation, with woody species such as Gossiaspp. poorly understood. At locations where G. acmenoideshas previously been found to accumulate\souts low levels of Mn, there may also be ion competition among similar divalent cations such as Ca2+, Mg2+ and Mn2+ in addition to less acidic soil conditions (Fernando et al., 2013, McLay et al., 2019, Bidwell et al., 2002). It is notable that high concentrations of Ca and Mg relative to Mn were reported in leaves of G. acmenoides (Bidwell et al. (2002).
In G. bidwillii , the behaviour of Mn in the oldest leaves resembled that of Ca, Mg and Na which remained high at all treatment levels but contrasted with that of K and P which decreased after maturity. However, in G. acmenoides , K was high in young leaves than in old leaves, whereas Ca, Mg, Na and Mn were higher in old leaves. The high concentrations of Mn, Ca, Mg and Na in old leaves of G. bidwillii and G. acmenoides could be attributed to phloem immobility of the aforementioned elements (Marschner, 2002, Graham et al. 1988) and vice versa for K in G . acmenoides andG. bidwillii . Moreover, this behaviour could be due to the similarities in divalent cations of Mn, Ca and Mg. Similar observation of high Mn in old leaves has been reported in G. bidwillii(Bidwell et al., 2002) and in other Mn hyperaccumulators includingPhytolacca americana (Xu et al., 2006), Macadamia integrifolia (Fernando et al., 2009a), G. fragrantissima(Abubakari et al. 2021) and in crop plants (Millikan, 1951). In contrast, G. grayi and G. shepherdii were reported to accumulate higher Mn concentrations in young leaves than in older leaves (Fernando et al., 2018). A previous report by Bidwell et al. (2002) of decreased Ca and Mg with an increase in Mn concentration in old leaves of G. bidwillii contradicts the findings of this study. The nutritional dynamics of plants as unusual as metal hyperaccumulators are yet to be fully understood, and clearly cannot be assumed to align with broader understanding of plant nutrition largely drawn from crop models (Marschner 2002).
The phytoavailability of Al and Mn are known to occur in soils of low pH (<5), however the pH of soils on which G. bidwillii was cultivated in this present study shows that the solubility of Al was low (>5), yet G. bidwillii was able to take up high amounts of Al in old leaves which qualifies it as an Al hyperaccumulator with concentration 6-fold higher than the Al hyperaccumulation threshold set at 3000 µg g-1 (Jansen et al., 2003, Jansen et al., 2001). Exceptionally high Al concentrations in old G. bidwillii leaves, even under Mn treatment, could be facilitated by organic anions involved in Mn transport (Bidwell et al., 2002). This suggests that G. bidwillii may be able to take up Al via an anion channel, a mechanism that appears to be a peculiar trait among Al tolerant species (Zhang et al., 2001, Ryan et al., 1997, Piñeros and Kochian, 2001, Kollmeier et al., 2001). It should be cautioned here that while these observations of Al over accumulation by G. bidwilliihave been made under experimental conditions, it has not been observed in the field, even on lateritic soils rich in Al (Fernando, Bidwell etc). The notable limit in uptake of Al in G. acmenoides even though its soil pH was suitably low for Al mobilisation for plant uptake, was most likely due to the very low soil Al concentrations. Furthermore, it has also been suggested that Al inhibits uptake of Ca and Mg in non- Al accumulators (Kochian et al., 2005, Ryan and Kochian, 1993, Rengel and Zhang, 2003), and this was found in old leaves ofG. acmenoides in this present study. Species within the Myrtaceae family have been listed to contain Al hyperaccumulators (Jansen et al., 2003, Jansen et al., 2001) and previous studies by Fernando et al. (2009b) have shown that other Gossia spp., includingG. hillii , G. inophloia , G. lewensis and G. macilwraithensis, can be Al hyperaccumulators after they were exposed to Al treatments.
The distribution patterns of Mn in leaves in wild G. acmenoidesand Mn-dosed G. bidwillii have been shown by XFM to be distinctly different (Figs. 3 and 4). Strong Mn distribution throughout young and old leaves in wild G. acmenoides , while highly concentrated at the apex and lamina of treated G. bidwillii suggest that Mn movement with the transpiration stream rather than against it. This indicates that, increasing transpiration rate throughout the leaves ofG. acmenoides , and towards the apex and lamina of G. bidwillii led to higher Mn accumulation in those parts of the studied species. Similar observations to that of G. bidwillii have been reported for the Mn hyperaccumulators Acanthopanax sciadophylloides (Memon et al., 1980) and G. fragrantissima(Abubakari et al., 2021a).
This study newly revealed the strong and facultative Mn hyperaccumulative trait in G. acmenoides and confirmed it inG. bidwillii , demonstrating both species to be strong Mn hyperaccumulators with concentrations in both young and old leaves well exceeding the Mn hyperaccumulation threshold. Laboratory based-XFM revealed distinct Mn distribution patterns in the leaves of G. acmenoides and G. bidwillii . Further work should be undertaken using synchrotron X-ray Florescence Microscopy with more precision and higher resolution to investigate Mn distribution at the cellular and subcellular levels in order to elaborate hypotheses for its metabolic pathways to be elucidated with genetic studies.