Comparable canopy and soil free-living nitrogen fixation rates in a lowland tropical forest
Graphical abstract
Introduction
Except for areas with high atmospheric nitrogen (N) deposition, biological N fixation (BNF) is the most important pathway for introducing ‘new’ N into unfertilized terrestrial ecosystems (Cleveland et al., 1999; Vitousek et al., 2013). Inert dinitrogen (N2) gas is reduced to ammonia during fixation by symbiotic or free-living N fixers (also called diazotrophs). Symbiotic diazotrophs, generally found in root nodules, exchange fixed N for carbon with their host plants, whereas hetero- or autotrophic bacteria or archaea freely inhabit and fix N in substrates such as water, soil, rocks, leaves, leaf litter and bryophytes (Dynarski and Houlton, 2018). The contribution of both these life strategies to the N cycle in tropical forests is thought to be substantial, estimated to range between 5.5 and 16 kg N ha−1 y−1 for symbiotic BNF, and between 0.1 and 60 kg N ha−1 y−1 for free-living BNF (Reed et al., 2011). Although many tropical forests have a high abundance of leguminous trees (Losos and Leigh, 2004), typically associated with symbiotic N fixers present in root nodules, the contribution of symbiotic BNF to the total BNF in tropical forests has been questioned because mature tropical forests are generally considered N rich compared to other nutrients, removing the need for trees to obtain N through symbiotic interactions with diazotrophs (Hedin et al., 2009). Based on mass-balance approaches and modelling Cleveland et al. (2010) showed that, after accounting for free-living N fixation and atmospheric N deposition, only modest inputs of N via symbiotic fixation were necessary to balance the N budget of a mature tropical forest in Amazonia. There is also increasing evidence that root nodulation and symbiotic N fixation is facultative and may decline to near zero in mature tropical forests (Menge et al., 2009; Barron et al., 2011; Batterman et al., 2013; Sullivan et al., 2014; Bauters et al., 2016), and therefore attention has been shifting towards the role of free-living BNF in the N cycle of tropical forests (Reed et al., 2011).
The availability of N, phosphorus (P) and even molybdenum - a necessary co-factor of many nitrogenases (Barron et al., 2009) -, in addition to humidity, have been shown to play an important role in determining free-living BNF rates (Reed et al., 2011; Wurzburger et al., 2012; Camenzind et al., 2018; Dynarski and Houlton, 2018; Van Langenhove et al., 2019). High rates of free-living BNF seem paradoxical (Hedin et al., 2009) in the face of the generally assumed N-rich and P-poor nature of mature tropical forests (Turner and Condron, 2013). However, because free-living BNF generally occurs in substrates decoupled from N conditions in deeper soils, such as the litter layer which is rich in C relative to N compared to decomposers (Menge et al., 2009), N inputs through free-living BNF can still be substantial in mature tropical forests (Hedin et al., 2009; Dynarski and Houlton, 2018). High rates of free-living BNF in tropical forest floor soil and litter have been reported in both Central (Reed et al., 2007; Barron et al., 2009; Černá et al., 2009; Cusack et al., 2009; Wurzburger et al., 2012) and South America (Matson et al., 2014), although markedly lower rates of free-living BNF were encountered in tropical forests in Mato Grosso State, Brazil (Wong, 2019), and in French Guiana (Van Langenhove et al., 2019), possibly related to the extremely low P availabilities there.
Beyond the forest floor, tropical rainforests possess extensive canopies, generally exceeding 30 m and regularly 45 m in height (Tao et al., 2016), representing a complex matrix of tree leaves and branches colonized by a diverse suite of animals and plants, such as bryophytes (including mosses, liverworts and hornworts), algae, lichens, fungi and vascular epiphytes (Nadkarni, 1994; Sillett and Antoine, 2004; Enloe et al., 2006; Nakamura et al., 2017). An additional component is canopy soil: accumulations of organic matter consisting of decomposing epiphytes, leaf litter, invertebrates, fungi and microorganisms found on branches and in tree junctions (Hietz et al., 2002; Nadkarni et al., 2002). Canopy soils display many similarities to tropical forest floor litter and soil (Vance and Nadkarni, 1990; Nadkarni et al., 2002; Cardelús et al., 2009). Microbial communities associated with these different canopy components do not have access to the soil N and may therefore fix N2 to meet their N requirements. Indeed, BNF by free-living diazotrophs has been found to occur on tropical leaf surfaces (Fürnkranz et al., 2008; Reed et al., 2013), on tropical bryophytes (Cusack et al., 2009) and in tropical canopy soils (Matson et al., 2014). One study even found that free-living BNF measured in canopy soils was higher than free-living BNF on the forest floor when comparisons were mass-based (Matson et al., 2014). Studies describing the role of vascular epiphytes in harbouring free-living BNF have reported variable results (Sengupta et al., 1981; Dighe et al., 1986; Bermudes and Benzing, 1991; Brighigna et al., 1992). Yet, to date, no study has aimed to quantify free-living BNF within the different canopy components simultaneously, nor attempted to estimate ecosystem-wide BNF including the canopy components.
Therefore, the aims of the present study were (1) to quantify free-living BNF rates of different canopy components (i.e. leaves, bryophytes, vascular epiphytes, canopy soil) and of forest floor (i.e. soil and leaf litter), and (2) to upscale these rates to the forest level to evaluate the relative importance of each component to the total amount of N fixed in an old-growth tropical lowland forest in French Guiana. By nature of the measurement technique, rates of free-living BNF are typically expressed on a per mass of substrate or a per area of substrate basis. However, to obtain ecosystem-wide (per hectare of forest) estimates of free-living BNF for the various canopy and soil components it is necessary to apply an appropriate scalar (Vitousek et al., 2013; Sullivan et al., 2014). In some instances this scalar is easily identified, as with forest floor soil for example. There, a measurement of BNF expressed on a per mass basis multiplied with the soil bulk density, corrected for the depth to which soil samples were taken, will yield an amount of N fixed per area of forest over a certain time period (see e.g. Matson et al., 2014). For other components, however, finding the appropriate scalar is not so straightforward and we here applied a combination of measured scalars for forest floor soil, leaf litter, canopy leaves and bryophytes while for canopy soils and epiphytes we applied scalars derived from a literature survey.
We hypothesized that because the canopy complexity creates niches for many free-living diazotrophs, free-living BNF occurring in the canopy contributes substantially to the total amount of N2 fixed and could make an important contribution to the N input at the ecosystem scale. We expect that because vascular epiphytes, bryophytes and canopy leaves make up a large part of the forest canopy they will each contribute more to the overall amount of N fixed through free-living BNF than canopy soils, which are much less prevalent.
Section snippets
Study area
The study was conducted at the Nouragues Nature Reserve, a primary rainforest site in French Guiana, situated 100 km inland from the Atlantic coast and south of the capital city Cayenne (4°2′ N, 52°40′ W). The site is located between 25 and 40 m above sea level, mean annual air temperature is 26 °C, and mean annual rainfall is 3000 mm (Bongers et al., 2001). The climate is characterized by a wet and a dry season due to the north/south movement of the Inter-Tropical Convergence Zone. The region
Results
Free-living BNF rates were highly variable, both within and among ecosystem components (Table 3). Across all ecosystem components, the lowest average ethylene production rate, as proxy for BNF, was 0.022 ± 0.009 (SE) nmol g−1 h−1, observed in soil, and the highest was 1.26 ± 0.41 nmol g−1 h−1, observed in canopy bryophytes (Table 3).
While there was no significant difference in either mass or area-based ethylene production rates between shaded and sunlit leaves, canopy bryophyte ethylene
Discussion
Our results demonstrated that in this mature lowland tropical forest ethylene production following acetylene addition, as a proxy for BNF, was an active process in canopy soil, on tree- and vascular epiphytic leaves, in bryophytes, in forest floor leaf litter and in topsoil. We found that 40% of the total ecosystem free-living BNF was carried out aboveground on tree trunks and within the canopy (Fig. 4), implying that aboveground free-living BNF constitutes a non-negligible contribution to this
Conclusion
Overall, rates of free-living BNF were low in our tropical forest site. Even so, 40% was carried out in the canopy and of all components both canopy bryophytes and canopy leaves showed the highest mass-based BNF rates and contributed most to total canopy BNF. The contribution made by vascular epiphytes and canopy soils was much smaller, at least in this forest. According to these results, future studies attempting to quantify ongoing BNF in lowland forest canopies will likely benefit from
CRediT authorship contribution statement
Leandro Van Langenhove: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization, Supervision. Thomas Depaepe: Formal analysis, Investigation, Writing - review & editing. Lore T. Verryckt: Conceptualization, Methodology, Investigation, Writing - review & editing, Visualization. Lucia Fuchslueger: Conceptualization, Investigation, Writing - review & editing, Supervision. Julian Donald: Investigation, Writing -
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This research was supported by the European Research Council Synergy grant ERC-2013-SyG. 610028-IMBALANCE-P, and the Ghent University Special Research Fund. The terrestrial LiDAR data were collected during a field campaign funded by the European Research Council Starting Grant 637643 (TREECLIMBERS). We thank the staff of the Nouragues station, managed by USR mixte LEEISA. (CNRS; Cayenne) for their hospitality and help in the field. The research station received support from Investissement
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2021, Science of the Total EnvironmentCitation Excerpt :However, more recent data suggest that these rates may be 10–20 times overestimated and point to BNF inputs in tropical forests of 1.2 kg N ha−1 yr−1 (Sullivan et al., 2014). Nevertheless, it has been estimated an ANF derived input of 2 kg N ha−1 yr−1 in lowland tropical forests in the French Guiana (Van Langenhove et al., 2020), and approximately 7–9 kg N ha−1 yr−1 in tropical and sub-tropical wet forests of Latin America (Reis et al., 2020). Our data suggest that for the Amazon forest site studied, the inputs of N through ANF in the phyllosphere (trees with more than 10 cm dbh, not considering understory species), litter and rhizospheric soil represents approximately 46% (38–59%, 95% confidence interval) of the BNF derived input estimated by Sullivan et al. (2014).