Fixation

Rates of nitrogen fixation were not measured in the sites sampled for this thesis. However, as biological nitrogen fixation (BNF) is the primary mechanism by which new nitrogen is conveyed to natural systems, the process warrants consideration in this review. Nitrogen fixation describes the conversion of inert N2 gas to bio-available N (Figure 2-2). Fixation occurs largely through biotic processes, although abiotic fixation also arises during lightning

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strike when N2 combines with oxygen to form NO and NO2 which subsequently precipitates as HNO3 (nitric acid). Approximately 10% of global reactive nitrogen (circa 12 Tg N y^-1) reaches the soil in this way (Price, et al., 1997). By contrast, BNF is estimated to contribute somewhere between 44 to 128 Tg N y^-1 to the global terrestrial input (Galloway, et al., 2004;

Vitousek, et al., 2013). In agricultural systems, BNF is either replaced by the application of artificial fertilisers or exploited through planting of N-fixing crops.

BNF breaks the triple bond of the N2 atom and combines N with H2 to form ammonia (NH3).

A wide range of phylogenetically diverse symbiotic and free-living bacteria, cyanobacteria and Achaea are able to fix atmospheric N, although legume-rhizobial symbiosis is perhaps the best-studied BNF mechanism. For the most part, symbiotic nitrogen fixing woody species are absent from late-successional temperate forests and rates of free-living fixation are low. As such, many climax communities in Europe and North America show signs of N limitation in the absence of anthropogenic inputs. However, tropical lowland forests (at least in South America and Africa) are often abundant in canopy legumes (Crews, 1999; Sprent, 2009; Raes, et al., 2013). Southeast Asian forests by contrast are dominated by Dipterocarpaceae (Slik, et al., 2003). With the exception of BNF in flooded rice fields (not considered in this review), our knowledge of the role that free-living N-fixers play at the ecosystem level is still

relatively limited. However, the few estimates of free-living fixation that have been

conducted within the humid tropics suggest that rates of N fixation through this mechanism may also be significant (Cusack, et al., 2009; Cleveland, et al., 2010; Reed, et al., 2011).

Consequently, high rates of N fixation are commonly surmised as the mechanism for N richness observed in many old-growth tropical forests.

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Figure 2-3: Summary of hypotheses in resolution of the “nitrogen paradox” in tropical soils.

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The supposition that N fixation is responsible for N richness presupposes that fixers continue to fix even where N is abundant within the wider ecosystem. BNF is an inherently costly activity and as such, obligate continuation is inconsistent with both theoretical expectation and physiological observations of the facultative nature of the process. Accordingly,

resolution of what has been termed the “nitrogen paradox” in tropical soils has sparked much recent debate (Vitousek, et al., 2002; Houlton, et al., 2008; Hedin, et al., 2009; Reed, et al., 2011) and a number of explanations have been offered to resolve this apparent incongruity.

Explanations largely fall into two categories, those that propose a form of obligatory fixation to acquire supplementary benefits, or those that refer to imperfect facultative fixation (Figure 2-3).

McKey (1994) proposed that leguminous plants, which have a higher N status than non-legumes, acquire supplementary benefits from over-fixation in N-rich tropical soils.

Similarly, N fixation may also be advantageous to plants that require additional N for N-rich herbivory defence mechanisms (Menge, et al., 2008). However, whilst over-fixation may occur in some soils (Pons, et al., 2007; Menge & Hedin, 2009), most field evidence points to facultative fixation of both symbiotic and free-living fixers (Crews, et al., 2000; Pons, et al., 2007; Barron, et al., 2009; Barron, et al., 2011). Enriched ¹⁵N isotopic signatures further confirm field observations that only a small number of legumes present appear to actively fix (Yoneyama, et al., 1993; Sprent, et al., 1996; Martinelli, et al., 1999; Ometto, et al., 2006;

Nardoto, et al., 2013).

The large diversity of symbiotic and free living fixers with the capacity for over-fixation in tropical soils may also offer an alternative explanation based on the insurance hypothesis of Yachi & Loreau (1999). This hypothesis proposes that declines in ecosystem function caused by environmental change are stabilised by microbial biodiversity (Yachi & Loreau,

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1999). Thus, the ability to activate N fixation in response to N limitation following

disturbance may ensure competitive advantage for individual species whilst at the same time decreasing ecosystem (i.e. N status) variability. Studies in disturbed soils support this mechanism of facultative fixation. For example, in Panama nodulation in canopy gaps

associated with low N availability and/or increased N demand from recovering vegetation was six times higher than in the surrounding mature forest matrix suggesting an ability to respond to disturbance with increased fixation (Barron, et al., 2011). Furthermore, the abundance (though not diversity) of N fixing trees persisted at similar concentrations over a

chronosequence of 300 years of forest development, however active fixation declined as demand for nitrogen decreased (Batterman, et al., 2013).

Another explanation for the high N status of tropical forests proposes that spatial variability in N availability may result in regions of high fixation despite overall N-richness. This suggests that where N availability is low, or C:N ratios are high, the demand for N at a local scale could keep BNF at an over-fixation level in the wider ecosystem. Hedin et al. (2009)

highlight the significance of vertical layering within tropical forests where persistent organic inputs to the litter layer and shallow rooting zone maintain N exigency. Under such

conditions, heterotrophic bacteria may continue to fix even where N is non-limiting in deeper soil horizons. Furthermore, epiphytic fixers, which are numerous in tropical forests, are disconnected from the soil matrix and therefore unable to access freely available N. This may result in an imperfect feedback mechanism where facultative down-regulation is blocked by an uncoupling of soil and canopy processes (Bentley, 1987; Cusack, et al., 2009).

Houlton et al. (2008) propose a somewhat controversial third explanation: that there is an advantage in maintaining N fixation because it increases competitive advantage for P.

Specifically, extracellular phosphatases responsible for the breakdown and availability of P

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are N-rich (circa. 15%-N) metabolites. Therefore, in P-limited environments, such as lowland tropical zones, N richness may be employed as a P acquisition strategy. Although soil

phosphatase activity is observed to be higher under leguminous plants (Keller, et al., 2013), N2 fixation does not necessarily translate to greater investment in extracellular phosphatase and alleviation of P limitation (Batterman, et al., 2013). Our understanding of the exact mechanisms for N richness within lowland tropical environments is at present incomplete.

For the most part, evidence points to the facultative nature of fixation in tropical forests.

However, a large diversity of fixers with capacity for over-fixation may insure against ecosystem decline in an environment characterised by an open cycle where nitrogen losses can be rapid following disturbance. When activities such as logging and burning result in the export of N through biomass removal, leaching, erosion and volatilisation, the capacity for BNF to supply new N to the soil is instrumental to forest recovery post-disturbance

(Batterman, et al., 2013; Barron, et al., 2011). In Panama, nitrogen fixing trees played a fundamental role in nitrogen accumulation over a relatively short time-scale with maximum rates of fixation being observed in 12 year old stands (Batterman, et al., 2013). However, decreased N demand, in parallel with a slowdown in biomass accumulation, decreased rates of fixation between 12 – 80 years to the point where 300 year old stands had similar rates of fixation to old growth forests. These observations raise an important point with respect to the secondary forests sampled for this study. Specifically, N limitation in early successional or highly disturbed forests is likely to result in high rates of nitrogen accumulation and

conservation illustrative of a closed N cycle. Whereas late-successional forests should have greater soil and plant nitrogen, and greater N losses indicative of a more open N cycle similar to that observed in old-growth tropical forests. Therefore, prior to determining the effect of forest conversion to alterative land uses, it is important to establish whether the secondary

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forests sampled for this thesis fall into the former category of a conservative and closed N cycle, or the latter category of open and leaky N cycle.