Plant source and soil interact to determine characteristics of dissolved organic matter leached into waterways from riparian leaf litter

Journal Pre-proofs Plant source and soil interact to determine characteristics of dissolved organic matter leached into waterways from riparian leaf litter Hannah M. Franklin, Anthony R. Carroll, Chenrong Chen, Paul Maxwell, Michele A. Burford PII: DOI: Reference:

S0048-9697(19)34521-8 https://doi.org/10.1016/j.scitotenv.2019.134530 STOTEN 134530

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Science of the Total Environment

Received Date: Revised Date: Accepted Date:

12 July 2019 12 September 2019 16 September 2019

Please cite this article as: H.M. Franklin, A.R. Carroll, C. Chen, P. Maxwell, M.A. Burford, Plant source and soil interact to determine characteristics of dissolved organic matter leached into waterways from riparian leaf litter, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134530

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Research Article

Plant source and soil interact to determine characteristics of dissolved organic matter leached into waterways from riparian leaf litter Authors Hannah M. Franklina, Anthony R. Carrollc,d, Chenrong Chena,b, Paul Maxwelle, Michele A. Burforda,b a

Australian Rivers Institute, Griffith University, Nathan, 4111, Brisbane, Queensland, Australia b

School of Environment and Sciences, Griffith University, Nathan, 4111, Brisbane, Queensland, Australia c Environmental

Futures Research Institute, Griffith University, Southport, 4222, Gold Coast,

Queensland, Australia d Griffith

Institute for Drug Discovery, Griffith University, Nathan 4111, Brisbane,

Queensland, Australia e

Healthy Land and Water, Brisbane City, 4111, Brisbane, Queensland, Australia

Details of corresponding Author: Hannah Franklin Address:

Australian Rivers Institute, Griffith University, Nathan, Queensland, 4111, Australia

ORCiD: http://orcid.org/0000-0003-1228-8243 Email: [email protected] Telephone: +61 7 3735 4370 Fax: +61 7 3735 7615 Mobile: +61 415 031 384 Author contributions: HF, CC, AC, FL, PM, MB conceived the ideas. HF undertook the technical work and data analysis. HF and MB wrote the majority of the manuscript, HF, CC, AC, FL, PM, MB, contributed to editing of the manuscript.

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Research Article

Plant source and soil interact to determine characteristics of dissolved organic matter leached into waterways from riparian leaf litter Abstract Wetting of leaf litter accumulated in riparian zones during rainfall events provides pulses of dissolved organic matter (DOM) to rivers. Restoring riparian vegetation aims to reduce sediment and nutrient transport into rivers, however DOM from leaf litter can stimulate phytoplankton growth and interfere with water treatment processes. Improved understanding of the loads and chemical composition of DOM leached from leaf litter of different plant species, and how subsequent leaching through soils affects DOM retention or transformation, is needed to predict the outcomes of riparian revegetation. To investigate this, we simulated rapid leaching of rainfall through the leaf litter of two riparian tree species with and without subsequent leaching through soil, comparing dissolved organic carbon (DOC) and nitrogen (DON) loads, and DOM chemical composition (via spectroscopic and novel NMRfingerprinting techniques). Plant source affected the load and composition of DOM leaching, with Eucalyptus tereticornis leaching more DOC than Casuarina cunninghamiana. Additionally, E. tereticornis DOM had a higher sugar, myo-inositol, benzoic acid, flavonoid and oxygenated aromatic content. More than 90% of leaf litter DOM was retained in the soil under simulated repeated heavy rainfall. The DOM chemistry of these species determined the total loads and changes in DOM composition leaching through soil. Less E. tereticornis DOM was retained by the soil than C. cunninghamiana DOM, with sugars, myo-inositol and amino acids being poorly retained compared to fatty acids and aromatic compounds. It also appears that DOM from E. tereticornis litter primed the soil, resulting in more DON being

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leached compared with bare soil. In comparison, C. cunninghamiana litter resulted in greater retention of DON, oxygenated aromatic compounds and the amino acid tryptophan. This study provides new information on how a range of DOM sources and transformations affect the DOM ultimately leached into waterways, key to developing improved models of DOM transformations in catchments. Keywords: humic substances; dissolved organic carbon; dissolved organic nitrogen; NMRfingerprinting; riparian restoration; rainfall events

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1. Introduction Terrestrially derived dissolved organic matter (DOM) represents an important energy source for microorganisms, algae and higher plants in freshwaters (Brett et al., 2017; Steinberg et al., 2006), influencing community structure and function (Berggren et al., 2010; Karlsson et al., 2015; Solomon et al., 2015). Conversely, some forms of DOM can inhibit cyanobacterial growth (Neilen et al., 2017), enhance the bioavailability of soil-derived nitrogen to freshwater and marine phytoplankton (Franklin et al., 2018; Garzon-Garcia et al., 2018), and increase the bioavailability of metals to aquatic organisms (Baken et al., 2011). Dissolved organic matter can also react with disinfectants, such as chlorine, during drinking water treatment forming disinfection by-products (Richardson and Postigo, 2015) linked to adverse effects on human health (Villanueva et al., 2007). Leaching of DOM during rainfall events, either directly from decaying vegetation or via soil leaching and runoff, are the dominant pathways through which terrestrial DOM enters rivers (Kaplan and Newbold, 1993). Land managers seek to restore riparian vegetation to protect these ecosystems and adjacent waterways from threats such as grazing, invasive plant species, fire and floods (Poff et al., 2011). Riparian vegetation can function to stabilize river banks and trap sediment and nutrients, reducing their transport from catchments into rivers (Dosskey et al., 2010). However, riparian vegetation is an important source of leached terrestrial DOM entering rivers and lakes, primarily through litterfall and subsequent DOM leaching during rain events (Meyer et al., 1998; Schlesinger and Melack, 1981; Yang et al., 2015). Readily soluble substances are leached from leaf tissues (then classed as DOM) at relatively high rates over the first 24-48 h after falling into waterways (Gessner et al. 1999; Gunnarsson et al. 1988) or when heavy rainfall results in water passing through or across leaf litter on the forest floor (Cleveland et al., 2015). DOM consists of a complex mixture of humic substances, proteins, amino acids and carbohydrates (Frimmel, 1998). DOM from different types of vegetation and 4

soil has highly variable composition which can influence its bioavailability and persistence in freshwater systems (Findlay and Sinsabaugh, 1999; Parr et al., 2015) and the types of disinfection by-products formed during water treatment (Williams et al., 2019). An improved understanding of the forms and loads of DOM produced from soils with different types of vegetation is needed to minimize any potential negative effects of riparian revegetation. The main controls on litter DOM leaching are: (1) the quantity and composition of organic matter, (2) the physical environment (e.g. temperature), (3) the microbial community colonizing the leaves, and (4) leaf fragmentation by physical processes or macroinvertebrate grazing (Chapin et al., 2002; Gessner et al., 1999). Quantifying and characterizing the contribution of DOM directly leached from leaves falling directly into streams and lakes has received the most attention (i.e. Bayarsaikhan et al., 2016; Neilen et al., 2017), while the load and composition of DOM generated during rainfall events from leaf litter within the riparian zone is less well understood. DOM leached from leaves decomposing in waterways is directly available to bacteria and phytoplankton and is rapidly transformed by photochemical and microbial processes (Neilen et al., 2019). Whereas DOM leaching from leaves on the soil surface is subject to physical, chemical and microbial processes as it moves across the soil surface or through soil prior to entering water. Tree species affects the quantity and composition of DOM leaching from leaf litter (Kiikkilä et al., 2013; Neilen et al., 2017; Wickland et al., 2007). For example, studies have shown that deciduous trees leach higher concentrations of DOM than coniferous species (Harris and Safford, 1996; Hongve et al., 2000; Nykvist, 1963). These differences have been attributed to a lower C:N ratio and lignin content of deciduous leaves compared with coniferous leaves, facilitating faster microbial decomposition (Melillo et al., 1982). Therefore, the type and quantity of vegetation adjacent to a stream can have a large effect on the concentration and composition of DOM delivered into waterways. Previous studies have found DOM from

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riparian vegetation can either promote (Berggren et al., 2010), suppress (Neilen et al., 2017; Solomon et al., 2015) or have little effect (Benner et al., 2001) on freshwater phytoplankton growth. These inconsistencies may reflect variation in the chemical composition of DOM leached from different plant sources and transformations of DOM that occur within riparian soil and upon entering rivers. On entering soil, DOM leached from riparian leaf litter may undergo physicochemical sorption by soil components (organic matter or clay particles), or uptake and transformation by microorganisms (incorporation into metabolites or mineralization to CO2) resulting in a smaller fraction leached through soil into waterways (Meyer et al., 1998). However, in some instances, litter-derived DOM is known to have a positive priming effect, whereby the addition of DOM stimulates microbial activity to access and release DOM from organic material stored in soil (Kalbitz et al., 2007). In other cases, inputs of labile DOM from litter have resulted in reduced DOM leaching from soil by stimulating preferential uptake of leaf and soil derived organic compounds, termed negative priming (Potthast et al., 2010). These processes depend on the surface charge, hydrophobicity and bioavailability of compounds in the DOM pool (Kiikkilä et al., 2013). Due to the complexities of DOM interactions with soil, the effect of transport of litter-derived DOM through soil on loads and composition leaching into waterways remains poorly understood. Therefore, improved characterization of DOM leached from soils vegetated with disparate tree species will help predict downstream ecosystem effects of riparian restoration. Surface runoff and subsurface leaching are both important processes potentially delivering DOM leached from leaf litter into rivers. However, this study focuses on understanding how transport through soil during subsurface leaching effects DOM leached from leaf litter, as the interaction with soil is likely to be more substantial during this leaching than surface runoff.

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Understanding the interaction of leaf litter DOM with soil is challenging due to difficulties in identifying and quantifying different forms of DOM. Previously, elemental and spectroscopic measures have been used to estimate broad features of DOM chemistry, and assess relative differences (Benner et al., 2001; Helms et al., 2008; Minor et al., 2014). More recently, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been adapted from use in natural products chemistry (i.e. Senadeera et al., 2017) in order to distinguish and quantify more specific functional and compound groups present in aquatic DOM (Hertkorn et al., 2016; Neilen et al., 2019; Zhang et al., 2014), termed here as NMR-fingerprinting techniques. In this study, we used a combination of elemental, spectrophotometric and NMR fingerprinting techniques (1H and 13C NMR, as well as 2-D NMR) to experimentally investigate the composition of DOM leached from two plant species found globally, and how this is transformed with leaching through soil. Principally this study aimed to: (1) quantitively compare the loads of dissolved organic carbon and nitrogen, and the chemical composition of DOM leached from leaf litter of two riparian plant species during rainfall events, and (2) investigate the interaction of leaf litter DOM with riparian soil, and the effect on DOM load and composition leached from soil. We hypothesized that the chemical composition of DOM leached from leaf litter during rainfall would be species-specific (others observed differences after 24 h leaching, e.g. Neilen et al., 2017) and that retention of DOM during transport through the soil profile would relate to the bioavailability and charge of the organic molecules present (Meyer et al., 1998). This study took place in subtropical, southeast Queensland, Australia where rainfall typically occurs as several large events during summer, therefore we also aimed to study the impact of repeated rainfall events on DOM leaching from litter and soil.

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2. Materials and methods 2.1 Study site and tree species description A leaching experiment was set up using intact riparian soil cores with and without leaf litter to investigate how plant source, and the simulated interaction of leaf litter with soil determine the quantity and composition of DOM leaching to rivers. Leaves and soil cores were collected from the North Pine catchment (area of 348 km2) in subtropical, southeast Queensland Australia. This catchment is dominated by natural forest with some cattle grazing (30%) (Olley et al., 2013). Natural vegetation cover was largely Eucalyptus-dominated open sclerophyll forest with some riparian closed-vine forest (Hodgkinson et al., 2006). The catchment receives an average annual rainfall of 1175 mm (Queensland Bureau of Meteorology, www.bom.gov.au), mainly in austral summer months (Hodgkinson et al., 2006). The majority (>60%) of suspended sediment in the North Pine River is generated by river bank erosion and less results from surface-soil erosion throughout the catchment (Olley et al., 2013). In the upper tributaries of the North Pine River large bands of metamorphic rock generate fast surface water runoff, especially where the landforms are steep, while lower in the catchment (including the area surrounding our study site) large alluvial deposits and moderate slopes enable water infiltration through soils to surface waters and groundwater recharge (Department of Environment and Science, Queensland, 2017). Soil cores were collected from a riparian area adjacent to the North Pine River (27°13'17.13"S, 152°50'25.27"E). The soil was classified as a hard pedal mottled-yellow-grey duplex soil, with a hard setting A horizon, A2 horizon conspic bleached, acid pedal mottled B horizon (Atlas of Australian Soils Queensland, http://www.asris.csiro.au; Paleustalf, Soil Survey Staff, 2014). Cores were collected from the river bank (~5 m from the wetted area) on the flat surface of a raised bank (2 m above the water height) from an area (~10 m2) with

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minimal vegetation or leaf litter cover. To determine soil texture, moisture, C and N concentrations and speciation, and the extent of soil variation within the collection area, triplicate additional cores were collected using a soil auger sampling at 0–10, 10–20 and 2030 cm depths. Soil moisture was measured gravimetrically on a subsample from each core (oven dried at 105°C for 48 h, detection limit 1.5%) and remaining soil was sieved to 2 mm for physical and nutrient analyses. Soil texture was determined using particle size analysis with the hydrometer method (Black, 1965, oven dried at 105°C for 48 h, detection limit 1%). Samples for C and N analyses were oven dried at 40°C for 48 h. Soil total N and C were analyzed by Dumas combustion, organic C by loss on ignition method (detection limits, 0.05%, ), and extractable ammonium (NH4+-N) and nitrate (NO3--N) by extraction with 2 mol L-1 potassium chloride (detection limits, 2 mg kg-1) (Rayment and Lyons, 2011). Sieved soil (<2 mm) was used to determine soil electrical conductivity and pH in a 1:5 (v/v) soil/water ratio. Leaf material was collected from two evergreen Australian native species commonly found along rivers and creeks in southeast Queensland, Australia, i.e. Eucalyptus tereticornis subsp. tereticornis (Queensland blue gum) and Casuarina cunninghamiana subsp. cunninghamiana (river she-oak) (Olley et al., 2014). Eucalyptus tereticornis is a fast-growing (up to 50 m tall) widespread forest tree of eastern Australia (Boland et al., 2006) and is a food source for the mammal species, the koala (Eldridge et al., 1993). Adult leaves are 8-22 cm long and 1-3.5 cm wide, tapering towards the petiole, dull or glossy, green in color, with notable oil glands are present (Fig. 1). Casuarina cunninghamiana is a pioneering nitrogen-fixing tree which occurs across much of eastern Australia, predominantly alongside and in riverbeds (Boland et al., 2006). Casuarina cunninghamiana is a slower growing species (up to 35 m) and has fine, waxy, needle-like dark green foliage. The foliage consists of drooping leaf-like twigs (6–9 mm long, 0.4–0.6 mm diam.) (Fig. 1). Teeth-like leaves (<0.5mm long) occur in rings of 8-6

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along the twigs, about 5 mm apart. Herein we refer to the C. cunninghamiana foliage as leaves, as these twigs are the principal photosynthetic apparatus of the tree and dominate the litter fall load (Clarke and Allaway, 1996). Both species loose foliage throughout the year, but losses peak in summer months, following periods of droughts or storm events (Burrows and Burrows, 1992; Clarke and Allaway, 1996). We chose E. tereticornis and C. cunninghamiana as focus species for this study as both are used widely in riparian revegetation projects in Southeast Queensland and Australia (Olley et al., 2014). They are also of genera that occur worldwide as forestry or invasive species, therefore information on the effects of these species on DOM leaching from riparian zones has board relevance. Eucalyptus tereticornis leaves were collected from trees (dbh 25-50 cm, 6.5-8 m tall) in a mixed native 10 ha plantation established in 2013. This was adjacent to the soil collection site (27°13'17.20"S 152°50'21.39"E). Casuarina cunninghamiana leaves were collected from trees which established on a gravel bar in the river bed adjacent to the plantation site following a flood in 2013 (dbh 31-39 cm, 6-9 m tall) (27°12'57.44"S 152°50'10.53"E). Green leaves were collected from five individual trees of each species seven days prior to use and were bulked together. To standardize the condition of leaves collected, we avoided fresh growth, dying leaves, and those with obvious insect or disease damage were discarded. In the laboratory, leaf material was rinsed for ~ 5 s under DI water to remove dust and insects, and patted dry. Large leaves were cut into 5-7 cm pieces, and oven dried at 50ºC for 48 h (following Neilen et al., 2017). Artificially created leaf litter was used instead of litter collected from the field in order to standardize the exposure and breakdown conditions between species, and increase the repeatability of the experiment. Foliar total N and C were analyzed by Dumas combustion (oven dried 48 h at 65°C, detection limits 0.1%) (Rayment and Lyons, 2011).

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Figure 1 Leaf litter of Eucalyptus tereticornis subsp. tereticornis (Queensland blue gum) (left) and Casuarina cunninghamiana subsp. cunninghamiana (river she-oak) (right) dried for 48 h at 50ºC. 2.2 Soil column and leaf litter leaching experimental setup Soil cores were collected on 13 and 14 November 2017 inside polyvinyl chloride PVC pipes (dimensions; 15 cm internal diam., 35 cm depth), which were pushed 30 cm into the ground and carefully excavated to maintain soil profile integrity. Cores were discarded when they became misshapen due to hitting large stones and or when soil became obviously compacted. PVC coated fiberglass mesh (0.5 mm pore size) was attached below pipes to contain soil. Cores were maintained for 2 weeks, in ambient light and temperature (mean daily 27ºC) but protected from rain by a plastic cover until the experiment commenced, to allow the microbial community to equilibrate and soil profile to settle after the disturbance caused during core extraction. Cores were placed on top of PVC funnels to allow for leachate collection. All plasticware was acid washed and rinsed with deionized water (DI water) prior to use. Comparison of DOC concentrations and NMR profiles of experimental blanks (DI

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water passed through the experimental set-up) and DI water confirmed that DOM did not leach from the PVC at detectable levels. There were five treatments in the leaching experiment, C. cunninghamiana and E. tereticornis litter (treatment ID’s: Euc and Cas), soil with C. cunninghamiana litter (S+Cas), soil with E. tereticornis litter (S+Euc) and soil only (S). To set up treatments Euc and Cas, 15 g of each species leaf litter was placed into five empty cores, capped at each end with mesh and placed onto funnels as per the soil cores (Euc and Cas). These treatments allowed characterization and comparison of loads and forms of DOM originating from leaf litter from each species. Five was deemed the maximum number of treatments possible to compare simultaneously in the experiment (therefore two plants species, with and without soil) whilst ensuring it was possible to filter the large amount of leachate generated on the same day as collection. Samples for DOM characterisation must be filtered rapidly as microbial activity may significantly reduce the concentration of low molecular weight organics (e.g. glucose and amino acids) within 24 h, even if samples are refrigerated (Brailsford et al., 2017). To investigate the interaction of leaf litter DOM with soil, 15 g of leaf litter of E. tereticornis and C. cunninghamiana was placed on top of five soil cores (S+Euc and S+Cas) and covered with PVC coated fiberglass mesh (0.5 mm pore size) to prevent wind-related losses and insect access. Five soil cores were left bare (S) to act as a control to account for DOM leaching from soil alone. Soil cores were randomly assigned to treatments. The addition of 15 g of leaf litter was based on field measurements of leaf litter standing stock during November 2017. All leaf litter was collected within three replicate 0.5 m2 quadrants placed randomly beneath the canopies of trees at six riparian sites for each species in the catchment, where cover was predominantly a monospecific stand of mature trees. To determine the standing stock litter was sorted to remove leaves of other species, twigs and animal matter (following Scarff and Westoby, 2006), dried for 48 h at 50ºC prior and weighed. Casuarina

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cunninghamiana had leaf litter with a dry weight of 823.5 ± 214.6 g m-2 (mean±SE) while E. tereticornis had 880.7 ± 68.2 g m-2. Litter was added to experimental cores at the same biomass for each species, equivalent to the mean of all field measurements (852.1 g m-2, therefore 15.0 g in 0.0176 m2 experimental cores). Leachates were collected on three occasions (at approximately 3 week intervals) after cores were irrigated with 0.6 L of DI water. The water was delivered in six 0.1 L aliquots poured through a watering can nozzle to simulate rainfall, one every 10 min during 1 h. The volume, rate and frequency of water added to the cores was designed to imitate subtropical rainfall events which occur during the summer months in this catchment (based on previous 20 years of available data from Dayboro Post Office station, Queensland Bureau of Meteorology, www.bom.gov.au). Soil was not pre-wet prior to the first leaching event (27 Nov 2017) so that when soils were wetted they simulated leaching losses during the first flushing event following the preceding dryer months. Two more leaching events took place 21 and 45 days later. Leachates were collected in acid-washed glass bottles, the volume measured and then filtered on the same day through 0.2 µm membrane filters (Millex-HV Filter, PVDF). An aliquot was put aside for spectral analyses the same day, another frozen at -20ºC prior to nutrient and carbon analyses, and the remainder frozen at -80ºC prior to freeze-drying for DOM analysis by NMR. 2.3 Chemical analyses of leachate DOM DOM was characterized through a combination of elemental analysis, optical characterization and proton (1H), carbon (13C) and 2-dimensional (2-D) nuclear magnetic resonance (NMR) spectroscopy. UV-visible absorption spectroscopy was selected as a representative method of optical characterization of DOM for use in this study, providing information about chromophoric DOM as a point of comparison to the NMR techniques used to provide more detailed structural information (Minor et al., 2014). Elemental analysis was used to determine 13

the concentration and load of dissolved organic carbon (DOC) and nitrogen (DON) delivered in each leaching event, together with the molar ratio of organic carbon to nitrogen (C:N). Total dissolved carbon (TDC), dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were determined by the combustion catalytic oxidation method (TOC Analyzer, TOCL with TNM-L measuring unit, Shimadzu, Kyoto, Japan, APHA/AWWA/WPCF, 2012, detection limit 0.05 mg L-1). Leachate concentrations of inorganic-N (NH4+−N and NOx: NO3-−N+NO2--N) were determined using colorimetric methods (APHA/AWWA/WPCF, 2012, detection limits, 0.002 NH4+-N; 0.001 NOx-N mg L-1). Dissolved organic nitrogen (DON) was calculated as DON = TDN – (NH4+−N + NO3—N). The proportions of the total carbon and nitrogen pools that were organic were calculated by diving the concentration of organic C/N by the total concentration or C/N. Loads of DOC and DON leached during each event (mg) were calculated by multiplying the concentration (mg L-1) by the leachate volume (L). Spectroscopic characteristics were measured on fresh leachate using ultraviolet (UV)-visible absorbance (Shimadzu UV-2450 Spectrophotometer, Kyoto, Japan) based on Helms et al., (2008). Absorbance spectra were recorded over the wavelength range of 290 to 750 nm, at 1 nm intervals, and a range of spectroscopic indices were used to infer DOM composition. Absorption coefficients (ag m-1) at 350 nm (a350) provided a proxy for lignin phenol concentration (Benner and Kaiser, 2011), and 440 nm (a440) as an indicator of gilvin content (yellow water color due to DOM) (McDonald et al., 2004). Specific UV absorbance at 254 nm (SUVA254; L mg-1 m-1) was an indicator of the aromaticity of DOC (Helms et al., 2008). The SR value, introduced by Helms et al., (2008) as a parameter negatively correlated with DOM molecular weight, is the ratio of the spectral slopes (S) obtained from the regions, 275 nm to 295 nm (S275-295) and 350 to 400 nm (S350-400). Each slope was calculated using linear regression of the log transformed spectral ranges (Yamashita et al., 2010).

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Leachates from the first two leaching events were also analyzed using solution-state 1H, 13C and 2-D NMR experiments to determine the principal chemical structures and features of compounds making up the DOM pool. Three replicate samples were analyzed for each treatment. Insufficient mass was leached from soil treatments on the final event to allow analysis. Freeze-dried DOM samples were dissolved in deuterated dimethyl sulfoxide (DMSO) and placed into sealed 3 mm diameter NMR tubes. NMR spectra were obtained at 25°C with a 500 MHz Bruker® Avance III HDX spectrometer equipped with a 5 mm tripleresonance cryoprobe (TCI). Details on the pulse program, scan number, relaxation delay, pulse width, spectral width, and spectra resolution are found in Hayton et al. (2017). The 1H and 13C chemical shifts and peak intensities were referenced to the residual proton resonance in d6-DMSO (Cambridge isotope Laboratory, Inc) at δH = 2.50 and δC = 39.52 ppm to identify compounds classes and their relative proportions. 13C

NMR techniques have been widely used to distinguish between and identify different

functional and compound groups present in DOM in natural waters (Nebbioso and Piccolo, 2013). However, in this study we followed a new approach recently adapted from use in natural products chemistry (e.g. Senadeera et al., 2017) to characterize DOM in leaf leachates (Neilen, 2018; Neilen et al, submitted). We used 2-D NMR techniques heteronuclear single quantum coherence NMR (HSQC-NMR) and correlation spectroscopy NMR (COSY-NMR) in addition to 1H and 13C, to provide more detailed assignment by determining the structural connectivity and proximity of specific functional groups. This combined approach provides a significant increase in sensitivity for identification of structural components with complex organic mixtures compared to solid-state NMR or 13C-NMR alone (Neilen 2018). Similar approaches have recently been used to characterize DOM in wetlands (Hertkorn et al., 2016) and lakes (Zhang et al., 2014).

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Peaks associated with seven specific compounds or compound classes were identified and quantified: fatty acids, myo-inositol, glucose, gallic acid, benzoic acid and derivatives, the amino acid, tryptophan and flavonoids (Table 3). Peaks were integrated and standardized to the residual proton signal in the d6-DMSO peak intensity to compare relative proportions quantitatively across samples. Relative loads of each compound group were calculated by multiplying the integral value by the leachate volume. Two regions associated with compound classes were also integrated and compared among treatments, amino acids (such as alanine, leucine, isoleucine, or valine) and oxygenated aromatic compounds, but could not be compared quantitatively to each other, or the specific compound classes identified.

2.4 Statistical analysis and calculations DOC and DON (concentration, total mass leached and percent composition as organic), spectroscopic and NMR relative proportion data were analyzed using general linear mixed effects models (nlme package in R, Pinheiro et al. 2017) followed by post-hoc comparisons of estimates (lsmeans package in R, Lenth 2016). Leaf litter/soil treatment type (S, S+Euc, S+Cas, Euc and Cas) and leaching occasion (Event 1-3: Day 0, Day 21 and Day 45) and their interactions were fixed factors in the model, while core number (ID) was included as a random effect to account for variance among the starting condition of individual cores, which are repeatedly sampled through time (see Crowder and Hand, 2017 for further details). When a significant Treatment*Time interaction occurred, the differences between treatment mean estimates for each leaching event were compared using Fisher’s LSD post-hoc tests. All data were analyzed for homogeneity of variance (Levene's test) and normal distribution (ShapiroWilk test) prior to analysis, and log or arcsine (percentage data as proportions) transformed when necessary to meet these assumptions. Principle components analysis (PCA) was conducted on data representing DOM elemental chemistry and composition (spectroscopic

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properties) from each leaching event, after transforming the data to meet the assumptions of normality, using the Euclidian distance function on standardized data. Pearson’s correlation coefficient was used to explore data for logical correlations between spectroscopic proxy values for DOM composition and NMR relative proportion data for compound classes identified. Loss or retention of DOC, DON (mass leached) and compounds of classes identified by NMR (relative mass) leached from leaf litter during transport through soil was calculated as a percentage relative to the mass leached from the litter as follows: 𝐿𝑜𝑠𝑠 𝑜𝑟 𝑎𝑑𝑑𝑖𝑡𝑖𝑜𝑛𝑎𝑙 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 (%) =

( 𝑀𝑎𝑠𝑠 𝑙𝑒𝑎𝑐ℎ𝑒𝑑 𝑠𝑜𝑖𝑙 𝑤𝑖𝑡ℎ 𝑙𝑖𝑡𝑡𝑒𝑟 ― 𝑀𝑎𝑠𝑠 𝑙𝑒𝑎𝑐ℎ𝑒𝑑 𝑠𝑜𝑖𝑙) 𝑋 100 𝑀𝑎𝑠𝑠 𝑙𝑒𝑎𝑐ℎ𝑒𝑑 𝑙𝑒𝑎𝑓 𝑙𝑖𝑡𝑡𝑒𝑟

Positive values in the range of 0-100% represent the portion of element/compound mass leached from litter that passed through the soil profile (loss). Positive values >100% indicate that when litter was present the mass leached was more than that leached in combination from soil and leaf litter alone, i.e. 200 % implies that leaching was equivalent to that from bare soil plus double the amount of input from litter. A value of 0% would occur when leaching from bare soil and soil with leaf litter are equal, i.e. all mass leached from litter was retained. Negative values represent additional retention of an element or compound when leaf litter is present, relative to the mass leached from the leaf litter, i.e. less mass leached when leaf litter was present compared with bare soil. The significance of mean loss/retention values was assessed as no overlap in the standard error with 0%. Statistics were performed in R version 3.0.1 (R Development Core Team, 2010, Vienna, Austria, http://www.r-project.org/).

3. Results 3.1 Riparian soil and leaf litter characteristics

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The soil used in this experiment was classed as a sandy loam (72.5% sand, 14.4% clay and 13.1% silt, soil profile average) (Table 1). Both soil total C (profile average of 1.45%, mostly as organic C) and N (0.12%) decreased down the profile and the soil C:N ratio was ~12.5. More soil extractable NO3- (16.8 mg L-1) was present than NH4+ (4.9 mg L-1) and changed little throughout the profile. Soil properties varied relatively little across the core collection area. The total C and N content of C. cunninghamiana leaf litter were 48.6±0.1% and 1.9±0.1% respectively, while E. tereticornis had 50.2±0.6% total C and 1.7±0.1% total N. The C:N ratio for E. tereticornis was 28.8±0.5 while for C. cunninghamiana it was 25.0±0.4. Foliar moisture content was 47.5±1.4% E. tereticornis (mean±SE) and 44.8±0.6% for C. cunninghamiana.

Table 1: Soil chemical and physical properties (mean±SE) in depth increments through the profile where the soil cores were collected for the leaching experiment. Depth increment in soil profile 0-0.1 m

0.1-0.2 m

0.2-0.3 m

Total carbon (%)

1.77 ±0.03

1.35 ± 0.03

1.24 ± 0.18

Total nitrogen (%)

0.14 ±0.01

0.11 ± 0.01

0.10 ± 0.01

Carbon: nitrogen ratio

12.4 ±0.6

12.4 ± 0.6

12.8 ± 0.2

Total organic carbon (%)

1.77 ±0.02

1.3 ± 0.02

1.19 ± 0.19

Ammonium (mg kg-1)

4.8 ±1.9

6.2 ± 1.9

3.8 ± 0.9

Nitrate (mg kg-1)

19.9 ±3.6

14.4 ± 3.6

16.0 ± 0.7

pH

6.1 ±0.2

5.7 ± 0.3

5.6 ± 0.2

Air dry moisture content (%)

18.6 ±1.4

21.7 ± 1.9

24.7 ± 2.1

Coarse sand (%)

22.0 ± 4.9

19.7 ± 4.9

23.2 ± 2.1

Fine sand (%)

50.8 ± 3.3

51.5 ± 3.3

50.3 ± 1.7

Silt (%)

13.3 ± 0.6

13.2 ± 0.6

13.0 ± 0.7

Clay (%)

13.9 ± 1.1

15.7 ± 1.1

13.5 ± 0.1

Particle size distribution

18

3.2 Experimental leaching of riparian soil with and without leaf litter 3.2.1 Loads and composition of DOM leached from Eucalyptus and Casuarina leaf litter E. tereticornis litter (Euc) leached more DOC mass than C. cunninghamiana (Cas) on the first and last leaching events, for the same biomass of leaves (Fig. 2a, Table 2). The mass of DOC leached increased from the first to second leaching event for both species. A relatively high proportion of the dissolved C pool was organic, with Euc leachate comprized of slightly more organic carbon (98.5±0.1%, mean±SE) than Cas (96.9±0.2%) (Table 2). Litter of both species leached similar amounts of DON (Figure 2b), but the Cas dissolved N pool consisted of more organic N (87.6±1.9%) than Euc (81.6±1.8%) on all sampling occasions (Table 2). Euc leachate had a higher DOC:DON ratio (255 ± 160) than Cas (122 ± 73) on all occasions and C:N ratios did not vary for each species over time (Table 2, Supplementary information, Table S1). The volume of water leached through litter of each species was similar and did not vary between leaching events (0.56±0.01 L per core) (Table 2). The chemical composition of DOM in leachate differed between species, and changed between leaching events (Fig. 3, Fig. 4, Fig. 5). Considering elemental chemistry and absorbance metrics as proxies for composition, the greatest variation in DOM leached was between plant species on the first event (no overlap in ordination space, Fig. 3). Euc leachate was darker colored than Cas (light orange vs light yellow, higher a440) and had higher aromaticity (SUVA254) and lignin-phenolic content (a350). On initial leaching, Euc produced more high molecular weight compounds (SR) than Cas, but in subsequent events, Cas leached more high molecular weight compounds. Euc leachate had higher lignin-phenolic content and color than Cas in the final leaching events.

19

NMR analysis was used to identify differences in the relative proportions of various classes of organic compounds that leached from each species (Table 3, Fig. 5). Only data from Days 0 and 21 are presented due to insufficient mass leached from some soil treatments on Day 45. Sugars (anomeric stereoisomers α- and β-glucose) and the metabolite of glucose, cyclic polyol, myo-inositol (cis-1,2,3,5-trans-4,6-cyclohexanehexol), were the specific compounds identified in highest relative proportions in leaf leachates, with significantly more produced from Euc than Cas (sugar; F4,20=4.9. p<0.01, myo-inositol; F4,20=8.1. p<0.001). Similar amounts of sugars and myo-inositol leached from Euc on both the first and second event, but significantly less of both compounds leached from Cas on the second event. (Treatment*Time; F4,20=4.3. p=0.03). Fatty acids were the next most concentrated compound class in leaf leachates (~5 fold less concentrated than sugars). More fatty acids leached from Euc on the second event compared with Cas. Compounds classed as gallic acid (3,4,5trihydroxybenzoic acid) and benzoic acid and derivatives (such as 4-hydroxybenzoic acid) and flavonoids were also present in leaf leachates, but at orders of magnitude less than sugars and myo-inositol. Benzoic acid and flavonoids were only detected in Euc leachate. Signals in the region representing oxygenated aromatic compounds (6.0-8.0 ppm) were more abundant and complex in Euc leachate compared with Cas on both the first and second leaching event (relative proportions of 1.6±0.2 and 0.4±0.01 respectively, Day 0, and 1.3±0.001 and 0.01±0.001 respectively, Day 21, Treatment effect; F4,20=24.6. p<0.001). Figure 4 presents example spectra from the first leaching event and all spectra can be found in Supplementary Information, Fig. S1. The amino acid profile (signals in region 0.9-1.20 ppm) was similar for both species’ leachate in the first leaching event, but amino acids were relatively more concentrated in Euc leachate than Cas in the second event (0.37±0.05 and 0.03±0.01 respectively, F4,20=2.9. p=0.04).

20

Figure 2 Mean±SE mass (mg per core) of dissolved organic carbon (a) and nitrogen (b) leached from Casuarina cunninghamiana litter (Cas), Eucalyptus tereticornis litter (Euc), bare soil (S), soil with E. tereticornis litter (S+Euc) and soil with C. cunninghamiana litter S+Cas). Data are for three separate leaching events, events two and three took place 21 and 45 days after the first leaching event (Day 0) respectively. Treatments which share a letter in common were not significantly different (p = 0.05) following post-hoc pairwise comparisons. Where no letters are present no significant differences were found. 21

1 2 3 4 5 6 7 8 9

Figure 3: Principle components analysis (PCA) ordination of DOM absorbance and concentration data for leaching events at 0, 21 and 45 days after the experiment started. Points represent leachate from individual cores (n=5) containing Casuarina cunninghamiana litter (Cas), Eucalyptus tereticornis litter (Euc), riparian soil (S), soil and E. tereticornis (S+Euc) or soil and C. cunninghamiana (S+Cas). Ellipses highlight the dispersion of selected species in ordination space (using the standard error of the weighted average of scores). Arrows show PCA loadings to describe the relationships between DOM absorbance and concentration variables for leaching events on days 0, 21 and 45. Abbreviations are: Specific UV absorbance at 254 nm, (SUVA254), dissolved organic nitrogen concentration (DON conc), dissolved organic carbon concentration (DOC conc), percent of the total dissolved nitrogen pool that was organic (% TDN organic), percent of the total dissolved carbon pool that was organic (%

TDC organic), the ratio of total dissolved organic carbon and nitrogen (DOC:DON), proxy for lignin phenol (a350), proxy for gilvin (a440), and the ratio of spectral slope (S) obtained from absorbance in the regions 275 nm to 295 nm (S275-295) and 350 to 400 nm (S350-400), inversely correlated with molecular weight (SR).

10 11 12

Table 2 Statistical results of general linear mixed models for dissolved organic carbon (DOC) and nitrogen (DON) metrics and the volume of water leached from cores. Significant differences from the control treatment are denoted by * - p > 0.05, ** - p > 0.01 and *** - p > 0.001.

22

DOC mass

DON mass

DOC concentration

DON concentration

Leachate volume

Proportion of C pool as organic F p

DF

F

p

F

P

F

p

F

p

F

p

Treatment

F (4,20)

201.9

***

6.44

**

135.6

***

9.2

***

180.4

***

47.7

Time

F (2,40)

16.9

***

3.9

25.2

***

3.2

21.6

***

1.7

Treatment*T ime

F (8,40)

20.9

***

8.7

25.5

***

7.9

8.9

***

6.5

***

***

***

Proportion of N pool as organic F

p

347.5

***

0.4 ***

10.9

DOC:DON ratio

13.2

***

3.3 ***

6.8

***

13

23

14 15 16 17 18 19 20

Table 3: 1H and 13C NMR (where available) chemical shift assignment of compounds and compound classes identified in DOM leach from leaf litter of Eucalyptus tereticornis (Euc), Casuarina cunninghamiana and (Cas), soil with Euc (S+Euc), soil with Cas and bare soil (S). Signal peak types are described as singlet (s), doublet (d), triplet (t) or doublet of doublets (dd) with associated coupling constants (J). Where multiple signals were used to identify a compound, the integral used for relative proportion and mass analysis is denoted in bold. X indicates the signal described was identified in one or more replicate of a treatment. Compound or compound class Region - Amino acids (such as Alanine, Leucine, Isoleucine, or Valine)

e.g. Alanine Fatty acids e.g.

myo-Inositol

β-Glucose (anomeric position) α- Glucose (anomeric position) Gallic Acid

δ1H (ppm) Multiple singlets in the region 0.9-1.20

δ13C (ppm)

0.83 (t, J = 7.85 Hz) 1.23 (s)

2.92 (t, J = 8.9 Hz) 3.36 (dd, J = 8.9, 9.4 Hz) 3.12 (dd, J = 2.7, 9.4 Hz) 72.4 3.73 (t, J = 2.7 Hz)

75.7 73.2

4.27 (d, J =7.2 Hz) 4.91 (d, J = 3.4 Hz) 6.92 (s)

964 91.6 109.2

Benzoic acid and derivatives 4-hydroxybenzoic acid

Indole NH proton of the amino acid tryptophan

Flavonoids

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

73.1

138.3 146.2 168.2 Region - oxygenated aromatic compounds

Presence or absence in each treatment Euc Cas S+Euc S+Cas S

Multiple signals (multiple types) in the region 6.0-8.0 6.88 (d, J = 8 Hz)

115.4

8.04 (d, J = 8 Hz)

130.4

X

9.35 (s)

12.52 (s) X

24

21 22 23 24

Figure 4: Comparison of selected 1H-NMR spectra for Casuarina cunninghamiana and Eucalyptus tereticornis litter and soil with and without this litter, from leaching event 1 (Day 0). All 1H-NMR spectra are shown in Supplementary Information.

25 25

26 27 28 29 30 31 32 33 34 35

Figure 5: Relative proportions of various organic compounds identified using unique signals in the 1H and 2-D NMR spectra of leachate from Casuarina cunninghamiana (Cas), Eucalyptus tereticornis litter (Euc) and riparian soil (Soil). Treatments which share a letter in common were not significantly different (p = 0.05) following post-hoc pairwise comparisons. Where no letters are present no significant differences were found. Data for soil with leaf litter (S+Euc and S+Cas) are not shown. The relative proportions of amino acids and oxygenated aromatic compounds are not shown as these are derived from integration of regions of the 1H NMR spectra and cannot be compared quantitively to compounds identified using unique signals.

36 37

26

38

3.2.2 Soil contributions to the DOM pool compared with leaf leachate alone

39

Water residence time in the soil profile was approximately 20-30 min. Soil treatments

40

produced less leachate volume than leaf litter alone (0.200±0.02 L , 0.270±0.03 L,

41

0.260±0.02 L for S, S+Euc and S+C respectively, Table 2), with up to 65% of water retained

42

by soil. The mean leachate volume decreased for each event in bare soil treatments but did

43

not vary for soil with leaf litter.

44

Significantly less DOC leached from bare soil (S) than leaf litter alone (Fig. 2a) and with

45

each leaching event (Table 2). Initially, more DON leached from S compared with Euc and

46

Cas (Fig 2.b), but the mass leached was similar in subsequent events. DON leached at higher

47

concentrations from S (6.0±2.4 mg L-1) than leaf litter treatments (0.8±0.08 mg L-1)

48

(Supplementary Information, Table S1), but due to differences in volume leached, overall

49

mass leached was similar in events 2 and 3.

50

DOM leaching from S had a different composition to leaf leachate alone (Fig. 3, no overlap

51

in ordination space). Based on elemental and absorbance metrics, soil DOM separated from

52

litter DOM along principal component axis 1. Soil DOM had a significantly a lower

53

proportion of organic compounds in both the C (94.6 ± 0.3%) and N pools (9.3±2.0%), and

54

lower C:N ratio (7.4±6.0) compared with leaf litter (Table 2 and Supplementary Information,

55

Table S1). On the second and third leaching events, compositional differences were also

56

driven by S leachates having relatively lower molecular weight (high SR values), lignin

57

phenol (a350) and gilvin (a440) content, compared with leaf litter (Fig. 3, and Supplementary

58

Information, Table S1). NMR analysis supported our finding that soil leached less phenolic

59

and aromatic compounds, compared with leaf litter. Soil also leached significantly less

60

glucose and myo-inositol than leaf litter (Fig. 5)

27

61

3.2.3 Interaction of leaf litter DOM with riparian soil

62

Over 90% of DOC leached from leaf litter was retained by the soil profile (Fig. 2a, i.e.

63

comparing soil+litter with soil alone, the difference equates to <10% of that leached from

64

litter). More DOC from Cas (97.0-99.6%) was retained than Euc (92.9-93.8%) and the

65

greatest retention by soil occurred for both species on the second leaching event. All DON

66

leached from litter of both species was retained on the first leaching event (Fig. 2b, i.e. no

67

additional DON leaching occurred when leaf litter was present). On subsequent events, for

68

S+Cas, less DON leached compared to S (bare soil), despite DON inputs leaching from Cas

69

increasing between the first and second leaching events. Euc leaching inputs stimulated

70

additional leaching of DON in S+Euc compared to S during the second and third leaching

71

events (DON mass leached equivalent to 4-5 times the input leaching from Euc). When litter

72

was present (S+Euc and S+Cas), the leachate C:N ratios were not different to the ratios in S

73

leachate. The organic portion of the leachate C (96.3%) and N pools (12.8%) increased in

74

S+Euc compared with bare soil.

75

DOM leached from soil with leaf litter present had distinct composition from other

76

treatments, but was compositionally more similar to soil leachate than leaf litter alone (Fig. 3,

77

S+Euc and S+Cas had more overlap in ordination space with S than Euc and Cas). On the

78

first leaching event, S+Cas leachate has similar elemental and absorbance profiles to S, while

79

S+Euc had intermediate composition between Euc and S only. During the second and third

80

leaching events, S+Euc leachate that had higher levels of color, lignin, phenol and gilvin, and

81

higher molecular weight (more like litter DOM) than S+Cas (Fig. 3, and Supplementary

82

Information, Table S1). In the second event, S+Cas had lower leachate color, lignin, phenol

83

and gilvin, and lower molecular weight compared with that of S. On the last leaching event,

84

leachates from soil plus leaf litter of both species had similarities to S, and were distinct from

85

leaf litter.

28

86

A portion of all identified organic compounds in Euc leachate passed through the soil profile

87

on the first and/or second leaching events, with the exception of the amino acid tryptophan

88

(Fig. 6, positive values represent the percent that passed through soil), whereas most organic

89

compounds leaching from Cas were completely retained by soil (Fig. 6, values negative or

90

not significantly different from 0). This was determined based on comparisons of the mass of

91

each compound leached when litter was present compared with bare soil, relative to the

92

amount that leached from the litter. Significant loss or retention occurred when error bars did

93

not overlap with 0%. Amino acids, sugars and myo-inositol leached from Euc had the lowest

94

retention in soil, ~10-30% passed through the soil profile. All gallic acid and flavonoids from

95

Euc were retained in the soil profile (mean loss/retention values not significantly different

96

from 0). On the second leaching event a portion of the sugar and myo-inositol in Cas leachate

97

passed through the soil profile. The amino acid tryptophan was not detected in Euc or Cas but

98

was present in all soil treatments (Table 3). The treatment with C. cunninghamiana litter on

99

soil (S+Cas) leached lower amounts of oxygenated aromatic compounds, the amino acid,

100

tryptophan and flavonoids than bare soil (S).

101

3.5 Relationship between measures of DOM composition

102

Of the elemental measures, DOC concentration was most highly correlated with

103

spectroscopic properties. DOC was significantly positively correlated with a350 (lignin derived

104

phenol), a440 (gilvin) (Fig. S1). Leachates of high lignin-phenol content (a350) and yellow color

105

(a440 , gilvin) were associated with relatively lower molecular weight (SR) . DOC and DON

106

concentrations were not correlated with any compounds identified by NMR. The organic

107

portion of the carbon pool had significant positive correlations with the relative proportions

108

of amino acids, myo-inositol, gallic acid and oxygenated aromatic compounds (Rho=0.47-

109

0.58), while the organic component of the dissolved N pool was positively correlated with

29

110

gallic acid and oxygenated aromatic compounds (Rho=0.51-0.53). No spectroscopic

111

properties were correlated with the relative proportions of compounds identified by NMR.

30

112 113 114 115

Figure 6: Loss (+ values) or additional retention (- values) of compounds of various classes leached from leaf litter during transport through soil. Values are a percent relative to the mass of each element of compound leached from the litter of each plant species. Data for amino acids and oxygenated aromatic compounds are derived from integration of multiple peaks in the regions of 0.9-1.20 and 6.0-8.0 ppm δ1H respectively, therefore represent multiple compounds within these groups.

116

31

4. Discussion Our study found over 90% of leaf litter-derived DOM was retained in the soil during simulated rainfall events, but the species of plant used affected the load and composition of organic compounds leaching from soil. The ability of soil to retain DOC from litter of these species was not affected by repeated leaching. The equivalent of 0.13 g DOC m-2 leached from soil without litter during simulated rainfall events of ~35 mm, while soil with E. tereticornis and C. cunninghamiana litter leached 0.24 and 0.15 g DOC m-2 respectively (mean of all events, scaled up from the mass leached from the soil core surface area of 0.02 m2). Annually, this would equate to 7.8 and 4.9 g m-2 y-1 leached through riparian zones sandy loam soils vegetated with E. tereticornis and C. cunninghamiana respectively, assuming all the regions 1175 mm annual rainfall was delivered in similar sized and sequenced events to those simulated in this study and leached through the top 30 cm of soil profile before entering the stream. Lower annual lateral flows of DOC through riparian soils of 1.5 and 2.3 g m-2 y-1 have been reported for boreal (Rasilo et al., 2015) and northern hardwood forests (Mcdowell and Likens, 1988) experiencing annual precipitation of ~650 and 1300 mm respectively. Estimated DOC flux through riparian soil in our study was more than double that of hardwood forests (Mcdowell and Likens, 1988), despite both systems receiving similar annual rainfall. In our study in a subtropical catchment, high annual DOC delivery may be driven by monsoonal rainfall events over the summer months which potentially release of large pulses of DOM from leaf litter (Zhou et al., 2016). Leaf litter exposed to ~35 mm rainfall (less than 1 minute of contact time with water) leached 3-5% of the mass of DOC compared with leaves of the same species soaking in water for 24 h (Neilen et al., 2017). In our study DOC leached from E. tereticornis litter at 1.8 mg g-1 dry leaf material and C. cunninghamiana 1.6 mg g-1 (average per leaching event), compared

32

with 61.4 and 37.1 mg g-1 for E. tereticornis and C. cunninghamiana respectively, when soaked for 24 h (Neilen et al., 2017). Only a small portion of this DOC leaching from litter during simulated rainfall passed through the soil profile (1-3% for C. cunninghamiana and 67% E. tereticornis). Other studies also report >90% reduction in DOC derived from vegetation during passage through upper soil horizons (Cronan, 1990; Mcdowell and Likens, 1988), showing that the amount of DOC leached through riparian soils into streams is controlled by differences in flow paths in the soil profile, i.e. contact time for retention in soil (Boyer et al., 2002). Our finding of high soil retention of litter-derived DOC suggests that hydrological flow paths within the North Pine catchment which divert water through the A horizon (top 30 cm) of riparian sandy loam soils will remove the majority of this DOC before it enters waterways. However, in the upper sub-catchments where water is channeled steeply towards rivers as overland flow (i.e. those with high bedrock cover), more riparian litterderived DOM may enter streams compared with low-gradient areas of alluvial deposit, where more water is likely to move through soil leading to greater retention, assuming the same amount of vegetation cover. Eucalyptus tereticornis litter leached a greater mass of DOC than C. cunninghamiana. E. tereticornis litter also leached more DOC in subsequent events whereas C. cunninghamiana did not. Neilen et al., (2017) found that Eucalypts tereticornis also leached more DOC than C. cunninghamiana (almost double) when soaked for 24 h, implying that this pattern continues through time. Casuarina cunninghamiana had lower foliar moisture content which has been found to drive litter degradability in other studies (Chadwick et al., 2003). This may explain in part the lower rate of DOC leaching by C. cunninghamiana compared with E. tereticornis. Larger DOC leaching losses are expected from leaf litter at lower C:N (Berg and Matzner, 2002) and lignin:N (Wieder et al., 2017) ratios due to increased bioavailability of organic compounds to microbes. However, in this study litter C:N ratios (E. tereticornis> C.

33

cunninghamiana) of both species was within the range for normal microbial activity (C:N ratio is 20–30) and is unlikely to be a factor driving differences in DOM release. E. tereticornis litter is likely to have lower lignin:N (11.0) than C. cunninghamiana (17.5) which may facilitate faster microbial breakdown (based on lignin values reported in Esslemont et al., 2007). This may explain the increase in DOC loss from E. tereticornis through time found in this study, compared to losses from C. cunninghamiana, which declined on the final leaching. Differences in initial chemical composition between leaves of different plant species is also likely to affect the rate and composition of DOM leaching. A previous study found C. cunninghamiana litter had greater percentage of lignin (~35%) and cellulose (~30%) and lower carbohydrate (~5%) and tannin (~5%) than several Eucalyptus species (E. largiflorens, E. viminallis and E. camaidulensis) (~20, 15, 10 and 10% respectively) (Esslemont et al., 2007). In our study, Eucalyptus tereticornis leached more gallic acid (a common plant phenolic compound) as well as sugars (carbohydrates) than C. cunninghamiana, which relate to differences in litter properties observed by Esslemont et al., (2007) and confirm findings by others that early stage DOM leaching is related to intrinsic litter properties (Kalbitz et al., 2006). Neilen et al., (2019) similarly identified tannin-related compounds (gallic acid and 4hydroxybenzoic acid) in leachate of E. tereticornis (24 h leaching) but not C. cunninghamiana. DOM lignin phenol content has been related to toxicity to cyanobacteria (Neilen et al., 2017), while, aromatic compounds, such as lignin and tannins (e.g. gallic acid) have been linked to disinfection by-product formation (trihalomethanes, Williams et al., 2019). Therefore, E. tereticornis litter in riparian zones may have a role in controlling harmful algal blooms, but increase formation of some disinfection by-products during drinking water treatment. The species-specific DOM structural information in this study

34

offers a first step in understanding the potentially complex effects of revegetating riparian zones with different tree species on downstream ecosystems and water treatment processes. A higher percentage of litter DOM from E. tereticornis passed through the soil profile compared with C. cunninghamiana, in particular glucose, myo-inositol and amino acids were retained poorly in soil. These compounds are more water soluble than fatty acids and the aromatic compounds identified in the leaf leachates, which may explain their comparatively poor retention. Sorption of DOM depends on the surface charge and hydrophobicity of the organic molecules and soils. Glucose molecules have no charge and there are no significant functional groups on the mineral surface of soil which soil can absorb sugars (Gunina and Kuzyakov, 2015), potentially leading to poor retention in soil. Glucose and other low molecular weight compounds, such as amino acids, delivered to rivers in runoff and subsurface leaching are likely to be highly bioavailable and rapidly removed from the DOM pool (Berggren et al., 2010) during rainfall events. This may explain why there are not typically identified in studies of freshwater DOM. The microbial uptake of sugars occurs within minutes in soils (Fischer et al., 2010). The presence of myo-inositol, a product of bacterial glucose metabolism (Reynolds, 2009) in all treatments implied that glucose is already being consumed rapidly during the litter and soil leaching processes. This concurs with findings of Neilen et al. (submitted). for the same plant species. Uptake and degradation by soil microbes is related to the biodegradability of litter-derived DOM, with more recalcitrant DOM which is produced by some tree species being less available to microbes (Wickland et al., 2007). However, in our study aromatic compounds in litter leachate tended to be retained in soil more than other compounds (e.g. glucose, fatty acids). This may be due to physicochemical rather than microbial processes, for example tannins may bind to proteins, or complex with metals and be retained in soil (Schmidt et al., 2012). Soil type is also likely to govern the transport and transformation of litter DOM during leaching and

35

should be considered in future studies. For example, in contrast to the sandy loam soil used in these experiments, less DOM may leach through clay soils due to low water infiltration and the high adsorption capacities of clay minerals (Deb and Shukla, 2011). E. tereticornis litter DOM not only leached through the profile to a greater extent than C. cunninghamiana, but also stimulated significant additional release of DON from the soil. Based on previous studies, it may be that inputs of tannins (and related phenolic compounds) and sugars leaching from litter into soil had a priming effect to drive microbial activity and increase short-term transformation of mineral N into organic forms which then leach (Castells et al., 2005; Gunina and Kuzyakov, 2015). Therefore, the higher tannin and sugar content of E. tereticornis leachate could explain the higher DON concentrations released from soil with E. tereticornis litter (S+Euc) compared with bare soil (S). Improved understanding of the organics C and N composition of DOM leached from vegetation and soil may help predict phytoplankton responses following rainfall events as organic nutrients have been found to modulate the bioavailability of sediment-bound inorganic nutrients (Garzon-Garcia et al., 2018). Despite the species differences in leaf litter/soil organic matter interactions in this study, the leached E. tereticornis DOM may not be delivered to streams at a greater rate than C. cunninghamiana DOM as E. tereticornis grows further from the wetted zone than C. cunninghamiana (which dominates river beds), potentially allowing more contact time with soil for these organic compounds to be retained (Cronan, 1990). Therefore, when revegetating catchments, planting E. tereticornis away from the river’s edge may minimize organic inputs from leaf litter leachate of this species in catchments where high organic load is of concern, e.g. drinking water catchments. 1H

and 2-D NMR methods complemented the use of traditional spectrophotometric measures

in this study. Broad differences between soil and leaf litter leachates in terms of the aromaticity and molecular weight of organic compounds measured using spectrophotometric

36

methods were confirmed by NMR. However, NMR provided additional structural resolution of the types of compounds present. For example, using 1H-NMR we quantified relative amounts of compounds that potentially have no chromophore, such as glucose, as well as fatty and amino acids (upfield of 3 ppm), therefore would not be visible using spectroscopic techniques. Alternative methods to quantify constituents of aquatic DOM, such as solid-state 13C

NMR (Liu et al., 2016; Schwede-thomas et al., 2005) and Fourier-Transform Ion

Cyclotron Resonance (FT-ICR) mass spectrometry (Abdulla et al., 2010; Hertkorn et al., 2016) are more common techniques to characterize complex DOM mixtures, however these are time consuming and often require additional sample concentration steps. The combined 1H

and 2-D NMR fingerprinting technique employed in this study (following Neilen et al.,

2019) allowed us to quantify compounds in samples from a complex experiment and with high time efficiency and signal resolution. This technique has promise for further development and use to study complex DOM dynamics in natural systems. For examples, in this study it facilitated understanding of the nature of the DOM interaction with soil. i.e. sugars and amino acids were leached through soil at higher loads than oxygenated aromatic compounds.

5. Conclusions Plant source affected the load and composition of DOM leaching during simulated rainfall on riparian soil, Eucalyptus tereticornis leached more DOC than C. cunninghamiana, and this DOM had a higher sugar, myo-inositol, benzoic acid, flavonoid and oxygenated aromatic content. More than 90% of leaf litter DOM was retained in soil under simulated repeated heavy rainfall, but the plant species modulated the load and composition of organic compounds leaching from soil. This was likely driven by differences in DOM chemistry. Less E. tereticornis DOM was retained than C. cunninghamiana, with sugars, myo-inositol

37

and amino acids having poor soil retention. Therefore, in rainfall events, both riparian species composition, and the extent of vertical and lateral water flow through soil are likely to influence the amount and composition of litter-derived DOM entering rivers. Eucalyptus tereticornis litter appeared to prime soil to leach additional DON compared with bare soil, while C. cunninghamiana litter stimulated additional retention of DON, oxygenated aromatic compounds and the amino acid tryptophan. Improved understanding how DOM from leaf litter of different plant species moves through the riparian zone and its role in priming soil processes will assist in restoration planning (i.e. which species to plant and at what position on river banks) and modelling of catchment organic carbon and N loads.

Acknowledgements The project was funded by the Australian Research Council under ARC Linkage Grant LP160100335 with MB, AC, FL, CC and PM as CIs. For assistance with field work and sample collection we thank Matthew Prentice and Brady Watt (Griffith University). For assistance with laboratory work we thank Ann Chuang, Jing Lu, and Vikki Lowe (Griffith University). Soil and plant chemical analysis was conducted by the Chemistry Centre, Department of Environment and Science, Queensland Government.

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Table 1: Soil chemical and physical properties (mean±SE) in depth increments through the profile where the soil cores were collected for the leaching experiment. Depth increment in soil profile 0-0.1 m

0.1-0.2 m

0.2-0.3 m

Total carbon (%)

1.77 ±0.03

1.35 ± 0.03

1.24 ± 0.18

Total nitrogen (%)

0.14 ±0.01

0.11 ± 0.01

0.10 ± 0.01

Carbon: nitrogen ratio

12.4 ±0.6

12.4 ± 0.6

12.8 ± 0.2

Total organic carbon (%)

1.77 ±0.02

1.3 ± 0.02

1.19 ± 0.19

Ammonium (mg kg-1)

4.8 ±1.9

6.2 ± 1.9

3.8 ± 0.9

Nitrate (mg kg-1)

19.9 ±3.6

14.4 ± 3.6

16.0 ± 0.7

pH

6.1 ±0.2

5.7 ± 0.3

5.6 ± 0.2

Air dry moisture content (%)

18.6 ±1.4

21.7 ± 1.9

24.7 ± 2.1

Coarse sand (%)

22.0 ± 4.9

19.7 ± 4.9

23.2 ± 2.1

Fine sand (%)

50.8 ± 3.3

51.5 ± 3.3

50.3 ± 1.7

Silt (%)

13.3 ± 0.6

13.2 ± 0.6

13.0 ± 0.7

Clay (%)

13.9 ± 1.1

15.7 ± 1.1

13.5 ± 0.1

Particle size distribution

Table 2 Statistical results of general linear mixed models for dissolved organic carbon (DOC) and nitrogen (DON) metrics and the volume of water leached from cores. Significant differences from the control treatment are denoted by * - p > 0.05, ** - p > 0.01 and *** - p > 0.001. DOC mass

DON mass

DOC concentration

DON concentration

Leachate volume

Proportion of C pool as organic F p

DF

F

p

F

P

F

p

F

p

F

p

Treatment

F (4,20)

201.9

***

6.44

**

135.6

***

9.2

***

180.4

***

47.7

Time

F (2,40)

16.9

***

3.9

25.2

***

3.2

21.6

***

1.7

Treatment*T ime

F (8,40)

20.9

***

8.7

25.5

***

7.9

8.9

***

6.5

***

***

45

***

***

Table 3: 1H and 13C NMR (where available) chemical shift assignment of compounds and compound classes identified in DOM leach from leaf litter of Eucalyptus tereticornis (Euc), Casuarina cunninghamiana and (Cas), soil with Euc (S+Euc), soil with Cas and bare soil (S). Signal peak types are described as singlet (s), doublet (d), triplet (t) or doublet of doublets (dd) with associated coupling constants (J). Where multiple signals were used to identify a compound, the integral used for relative proportion and mass analysis is denoted in bold. X indicates the signal described was identified in one or more replicate of a treatment. Compound or compound class Region - Amino acids (such as Alanine, Leucine, Isoleucine, or Valine)

e.g. Alanine Fatty acids e.g.

myo-Inositol

β-Glucose (anomeric position) α- Glucose (anomeric position) Gallic Acid

δ1H (ppm) Multiple singlets in the region 0.9-1.20

δ13C (ppm)

0.83 (t, J = 7.85 Hz) 1.23 (s)

2.92 (t, J = 8.9 Hz) 3.36 (dd, J = 8.9, 9.4 Hz) 3.12 (dd, J = 2.7, 9.4 Hz) 72.4 3.73 (t, J = 2.7 Hz)

75.7 73.2

4.27 (d, J =7.2 Hz) 4.91 (d, J = 3.4 Hz) 6.92 (s)

964 91.6 109.2

Benzoic acid and derivatives 4-hydroxybenzoic acid

Indole NH proton of the amino acid tryptophan

Flavonoids

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

73.1

138.3 146.2 168.2 Region - oxygenated aromatic compounds

Presence or absence in each treatment Euc Cas S+Euc S+Cas S

Multiple signals (multiple types) in the region 6.0-8.0 6.88 (d, J = 8 Hz)

115.4

8.04 (d, J = 8 Hz)

130.4

X

9.35 (s)

12.52 (s) X

46

Highlights 

Leaf litter source affected the load and composition of leached DOM



Over 90% of DOC leached from litter was retained by soil in simulated heavy rainfall

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Leaf leachate DOM profile determined the loads and composition leaching from soil

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Casuarina litter primed DON retention in soil, while Eucalyptus enhanced losses

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NMR fingerprinting quantified sugars, amino and fatty acids leaching to waterways

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