Assessing branched tetraether lipids as tracers of soil organic carbon 1 transport through the Carminowe Creek catchment (southwest 2 England)

. Soils represent the largest reservoir of organic carbon (OC) on land. Upon mobilisation, this OC is either returned 11 to the atmosphere as carbon dioxide (CO 2 ), or transported and ultimately locked into (marine) sediments, where it will act as 12 a long-term sink of atmospheric CO 2 . These fluxes of soil OC are, however, difficult to evaluate, mostly due to the lack of a 13 soil-specific tracer. In this study, a suite of branched glycerol dialkyl glycerol tetraethers (brGDGTs), which are membrane 14 lipids of soil bacteria, is tested as specific tracers for soil OC from source (soils under arable land, ley, grassland and 15 woodland) to sink (Lake Loe Pool sediments) considering a small catchment located in southwest England (i.e. Carminowe 16 Creek draining into Lake Loe Pool). The analysis of brGDGTs in catchment soils reveals that their distribution is not 17 significantly different across different land use types ( p > 0.05), and thus does not allow tracing land use-specific soil 18 contributions to Lake Loe Pool sediments. Furthermore, the significantly higher contribution of 6-methyl brGDGT isomers 19 in creek sediments (isomerization ratio (IR) = 0.48 ± 0.10; mean ± s.d., standard deviation; p < 0.05) compared to that in 20 catchment soils (IR = 0.28 ± 0.11) indicates that the initial soil signal is substantially altered by brGDGT produced in situ . Similarly, the riverine brGDGT signal appears to be overwritten by lacustrine brGDGTs in the lake sedimentary record, 22 indicated by remarkably lower Methylation of Branched Tetraethers (MBT' 5ME = 0.46 ± 0.02 in creek bed sediment and 0.38 23 ± 0.01 in lake core sediment; p < 0.05) and higher Degree of Cyclisation (DC = 0.23 ± 0.02 in creek bed sediment and 0.32 ± 24 0.08 in lake core sediment). Thus, in this small catchment, brGDGTs do not allow us to trace soil OC transport. Nevertheless, the downcore changes in the degree of cyclisation and the abundance of isoprenoid GDGTs produced by 26 methanogens in the Lake Loe Pool sediment do reflect local environmental conditions over the past 100 and have 27 recorded the eutrophication history of the lake.

4 by arable land and temporary grasslands (ley), which are under rotation. The steeper hillslopes are under permanent 119 grassland, and riparian woodland covers the areas near the creek. For this study, 74 surface soil samples (0-15 cm) were 120 collected along 14 hillslope transects, including 31 arable land sites, 14 permanent grassland sites, 24 temporary grassland 121 (ley) sites and 5 woodland sites (Fig. 2). Riverbed sediments were collected at three locations along each of the two 122 tributaries (upstream, midstream and downstream), and one more at the joint outlet. A 50 cm long sediment core was taken 123 in the lake, about 150 m away from the joint outlet. The lake core has been dated by the activity of Caesium-137 ( 137 Cs), and 124 it covers the last 100 years (Glendell et al., 2018). 125

Bulk soil properties 126
Total carbon contents were reported by Glendell et al. (2018). Soil pH was measured in this study using a pH meter in a soil 127 to water ratio of 1:5 (w:v) after shaking for two hours. 128

GDGT extraction and analysis 129
In total, 74 soil samples, 7 creek bed sediment and 25 lake core sediment samples were analysed for GDGTs. First, 5-7 g of 130 the soils or 3-5 g of the sediments were freeze dried and homogenized, after which they were extracted three times with 131 dichloromethane (DCM) : MeOH (9 : 1, v/v) using an accelerated solvent extractor (ASE 350, Dionex TM ) at 100 °C and 7.7 132 × 10 6 Pa to obtain a total lipid extract (TLE). After addition of a known amount of C46 GDGT internal standard (Huguet et 133 al., 2006), the TLEs were dried under a N2 stream, and then separated into apolar and polar fractions by passing them over 134 an activated Al2O3 column using hexane : DCM (9 : 1, v/v) and DCM : MeOH (1 : 1, v/v) respectively. The polar fraction, 135 which contains the GDGTs, was evaporated to dryness under a gentle N2 stream. After this, the samples were prepared for 136 further analysis by re-dissolving them in a hexane : isopropanol (99 : 1, v/v) mixture, and filtration through a 0. (1) 155 5 The degree of methylation (MBT'5ME) and relative abundances of tetra-, penta-, and hexamethylated brGDGTs were 156 The isomerization ratio (IR) is the ratio between penta-and hexamethylated 6-methyl brGDGTs and the total amount of both

Statistical analysis and data visualization 167
The statistical analysis and data visualization were undertaken in R programming (version 3.5.2) (R Core Team, 2018). 168 Differences in the concentration of brGDGTs and brGDGT-based proxies between different land use types (i.e. arable land, 169 grassland, ley and woodland), creek bed and lake core sediments were examined by one-way nested ANOVA under 170 generalized linear model (GLM) followed by post-hoc analysis (Tukey HSD (honest significant difference) test), and were 171 performed with package 'car', 'carData' and 'agricolae'. Differences were considered to be significant at level of p < 0.05. 172 To show how close our sample mean is to the population mean, standard deviation is used (mean ± s.d.). To examine 173 whether brGDGT signatures could distinguish soil OC derived from different land use types, principal component analysis 174 (PCA) was performed with package 'FactoMineR' and 'factoextra'. The box plot and scatter plots were carried out with 175 package 'ggplot2'. 176 3 Results 177

BrGDGTs in creek bed sediments 198
All brGDGT compounds were detected in creek bed sediments, except for in the upstream site from north catchment, where 199 brGDGT-IIIc' was below detection limit. The brGDGTs in creek bed sediments were dominated by pentamethylated 200 brGDGTs (45.0 ± 0.7%), followed by tetramethylated brGDGTs (30.1 ± 4.5%), and hexamethylated brGDGTs (24.9 ± 201 4.7%) ( Table 1). The C-normalized concentration of brGDGTs in creek bed sediments was 34.7 ± 17.4 g g -1 C on average 202 ( Fig. 3a; Table 1), where the concentration increased from 32.7 g g -1 C to 57.0 g g -1 C downstream in north catchment, 203 and from 14.3 g g -1 C to 25.2 g g -1 C downstream in south catchment, reaching a maximum value of 59.3 g g -1 C at the 204 outlet (Fig. 5a). The concentration of brGDGTs in creek bed sediments was higher than that in soils under any land use 205 types, except for woodland (9.6 ± 4.9 g g -1 C; Fig. 3a; Table 1). 206 The BIT values for creek sediments were on average 0.90 ± 0.06 ( Fig. 3b; Table 1). The MBT'5ME was relatively constant 207 between 0.44 and 0.49, with an average of 0.46 ± 0.02. The DC ranged from 0.21 to 0.25 in the creek sediments with an 208 average of 0.23 ± 0.02 ( Fig. 3e; Table 1). The IR was relatively invariable with an average of 0.48 ± 0.10 ( Fig. 3e; Table 1). 209 The brGDGT-based proxies for creek bed sediments were similar to those for soils, except for the IR, which was higher than 210 that in soils under any land use types (0.28 ± 0.11; Fig. 3; Table 1). 211

BrGDGTs in Lake Loe Pool sediment core 212
All brGDGTs were detected in the lake sediment core, except at 20 cm depth, where brGDGT-IIIc' was below the detection 213 limit. The brGDGTs in the lake sediments were mainly dominated by pentamethylated brGDGTs (50.2 ± 1.8%), followed by 214 tetramethylated brGDGTs (28.9 ± 0.7%), and hexamethylated brGDGTs (21.0 ± 1.4%; Table 1). The amount of brGDGTs in 215 lake core sediment ranged from 19.9 to 48.0 g g -1 C ( Fig. 3a; Table 1). The brGDGT concentration in the surface sediment 216 (0-2 cm), of 37.7 g g -1 C, which was about 1.6 times lower than that in the creek sediment at the outlet (Fig. 5a), increased 217 to a maximum of 48.0 g g -1 C around 11 cm depth, and then decreased to a minimum of 19.9 g g -1 C at 23 cm depth (Fig.  218 6b). The concentration of GDGT-0 ranged between 9.0 g g -1 C and 27.1 g g -1 C with an average of 17.4 ± 6.0 g g -1 C, 219 concentration of crenarchaeol ranged from 0.6 g g -1 C to 1.4 g g -1 C with an average of 1.0 ± 0.2 g g -1 C in the lake 220 sediment core. In general, the concentration of brGDGTs in lake core (34.0 ± 8.7 g g -1 C; Table 1) was similar with that in 221 river and in woodland, while it was significantly higher than the brGDGTs in soils except for the woodland (9.6 ± 4.9 g g -1 222 C; p < 0.05; Fig. 3a; Table 1). 223 The BIT values for the lake sediment core were rather uniform, varying between 0.95 and 0.97 (Fig.3b). Similarly, the 224 values of MBT'5ME along the lake core ranged only between 0.36 and 0.39. The MBT'5ME of 0.37 for the lake surface 225 sediment was significantly lower than that in creek bed sediment (0.46 ± 0.02; p < 0.05; Fig 3c; Fig. 5b). Conversely, the DC 226 in the lake surface sediment was 0.39, which was significantly higher than that in creek bed sediment (0.23 ± 0.02; p < 0.05; 7 Fig. 3d; Fig. 5b). The average value of DC for the lake core sediments was 0.32 ± 0.08. The DC increased from the surface 228 to a maximum value (0.44) at around 10 cm depth, and then decreased with slight fluctuations to 0.22 at 43 cm depth (Fig.  229 6c). The IR was constant downcore (0.32 ± 0.01 on average; Fig. 3e; Table 1) and was significantly lower than that in creek 230 increase from the presumably better aerated soils at the hilltops towards the wetter soils closer to the creek (Fig. A1). The 277 increase is > 0.3 for Transects 1 and 8, but also Transects 2, 3, and 7 show an increase in BIT values downslope, albeit to a 278 smaller degree (0.17, 0.19, and 0.04, respectively; Table A1 Interestingly, the IR is also significantly higher in soils along four transects in north catchment (all > 0.36 averagely for 287 Transects 1, 2, 7, and 8) compared to the average IR value for the rest of the transects in the entire catchment (0.24 ± 0.09; p 288 < 0.05). The majority of the sites with higher IR are in cropland, except for those in the Transect-1, which is under grassland 289 (Fig. A2). Although a relative increase in 6-methyl brGDGTs has been linked to higher soil pH in the global soil dataset (De 290 Jonge et al., 2014b), this relation is not so strong in the soils from the Carminowe creek catchment (r 2 = 0.36, p < 0.001), 291 likely due to the relatively minor range and variation in soil pH (from 5.4 ± 0.3 to 6.6 ± 0.1). Nevertheless, the soils with 292 high IR values in the north catchment also have pH values > 6.0 with an average value of 6.6 ± 0.1. 293

Tracing brGDGTs from soils to creek bed sediments 294
Based on the similar brGDGT signatures for soils under different land use types, these compounds cannot be used to trace 295 back the exact source of the soil OC after mobilisation and transport throughout the catchment. However, the concentration 296 and general soil signature of the brGDGTs can be compared with those in creek bed sediments to trace the transfer of OC 297 from the soils into the creeks. The C-normalized concentration of brGDGTs in the creek sediments is higher than that in 298 most of the soils (34.7 ± 17.4 g g -1 C and 9.6 ± 4.9 g g -1 C respectively), except for those in the woodland soils at the 299 riverbanks (37.6 ± 11.0 g g -1 C; Table 1). Thus, purely based on the concentration, this suggests that brGDGTs in the creek 300 would be primarily derived from the woodland, which also appeared to be the main source of n-alkanes in creek bed 301 sediment (Glendell et al., 2018). However, when looking at the relative distribution of the brGDGTs, the percentage of 302 hexamethylated brGDGTs in creek sediments is higher than that in soils (24.9 ± 1.8% and 10.9 ± 0.3%, respectively), 303 whereas the percentage of tetramethylated brGDGTs is lower than in soils (30.1 ± 1.7% and 39.7 ± 0.6%, respectively; Table  304 1). Furthermore, brGDGTs in creek sediments have a significantly higher IR (i.e. 0.48 ± 0.04) than soils under any of the 305 land use types (0.28 ± 0.01 on average in the catchment; p < 0.05; Fig. 3e; Table 1). This is clearly reflected in the PCA, 306 which separates the creek sediments from both the soils and lake sediments on PC2 that is associated with the IR (Fig. 4e). 307 The higher IR in the creek bed sediments can be explained by a contribution of aquatically (i.e. in situ) produced 6-methyl 308 brGDGTs. Similar contributions of 6-methyl brGDGTs, and thus higher IR, were also observed in suspended particulate 309 matters from the Yenisei River (De Jonge et al., 2014a), and upstream of the Iron Gates in the Danube River, where the 310 higher IR was coupled to in-river production facilitated by the lower flow velocity and decreased turbidity of the river water 311 (Freymond et al., 2017). Hence, the significantly higher IR in combination with the higher C-normalized concentrations of 312 brGDGTs in the Carminowe creek sediments suggests that the brGDGT signal is mainly aquatic. 313 In attempt to further prove the riverine in situ production of brGDGTs, we roughly estimate the minimum amount of 6-314 methyl brGDGTs that needs to be produced in the creek in order to reach the higher IR. We hereby assume that the 315 brGDGTs derived from woodland soils are completely transferred into creek without any degradation. Thus, the 316 concentration of 6-methyl brGDGTs in the creek sediments [6-me creek] resembles the sum of the average concentration of 6-317 methyl brGDGTs in woodland soils [6-me woodland] and those produced in situ [6-me in situ]. The minimum amount of 6-methyl 318 brGDGTs produced in situ can then be calculated using the brGDGT-concentration-weighed IR for creek sediments (IR creek 319 = 0.47) and the following equation (Eq. 8) (8) 321 Solving this equation results in a minimum amount of 7.4 g g -1 C 6-methyl brGDGTs that needs to be additionally 322 produced in the creek to reach the higher IR. This accounts for 65% of the total amount of 6-methyl brGDGTs in the creek 323 bed sediment that we measured. Considering a mixture of all soils rather than only woodland as source for soil-derived 324 brGDGTs in the creek results in the in situ production of 9.3 g g -1 C 6-methyl brGDGTs, corresponding to 81% of the 6-325 methyl brGDGT pool in the creek bed sediments. This implies that the initial soil brGDGT signal is rapidly overprinted by a 326 riverine in situ signal upon entering the creek. Only the IR for the downstream site in the northern creek approaches that of 327 the adjacent soil (IR = 0.30 in the creek bed sediment and 0.38 ± 0.07 for Transect-7; Fig. A2), and may be explained by its 328 use as arable land (Fig. 5a), which involves regular ploughing and subsequent soil mobilisation and implies a temporary, 329 local overprint.

Sources of brGDGTs in the sediments of Lake Loe Pool 341
In theory, rivers would transport soil-derived OC together with any aquatic OC produced along the way. Once discharged, in 342 this case into a lake, the OC would settle and then be buried into the sediments where it would act as a long-term sink of OC. 343 However, the soil brGDGT signal cannot be recognized in the sediments from Loe Pool since it is already lost upon entering 344 the Carminowe creek. Indeed, the PCA of the relative distributions of brGDGTs indicates that lake sediments plot 345 completely separated from both the soils and creek sediments, mostly due to a higher relative abundance of GDGT-IIIa (Fig.  346   4a, b). As a result, the MBT'5ME is significantly lower in Loe Pool sediments (0.38 ± 0.00) compared to in the creek bed 347 10 sediments (0.46 ± 0.01; p < 0.05) and soils (0.48 ± 0.01; p < 0.05; Fig. 5b; Table 1). Furthermore, the DC is significantly 348 higher in lake sediments than in both soil and creek bed sediments (0.32 ± 0.02, 0.23 ± 0.01 and 0.23 ± 0.01, respectively; p 349 < 0.05; Fig. 3d; Table 1). The distinct brGDGT signature of the lake sediments suggests that brGDGTs in the lake again are 350 significantly altered compared to those in the soils and creek sediments. This implies that the riverine brGDGT signal is 351 either replaced or overwritten in the lake. there are no generally recognized indicators (yet) to identify lacustrine brGDGT production, although several studies 355 reported a "cold bias" while attempting to reconstruct the mean air temperature (MAT) based on brGDGTs in lake sediments 356 using a soil-based transfer function (Tierney et al., 2010). In a study on East African lakes, this cold bias was linked to a 357 large in situ contribution of brGDGT-IIIa (Tierney et al., 2010), similar to in Loe Pool. However, the East African lake 358 dataset was generated using the 'old' chromatography method that does not separate 5-methyl and 6-methyl brGDGTs. A 359 recent study that has re-analysed the East African Lake dataset indicates that the presumed contribution of GDGT-IIIa 360 mainly consists of brGDGT-IIIa' (Russell et al., 2018), which is less prominent in lake Loe Pool. Although the identity of 361 brGDGT-producer(s) in lakes still remain(s) elusive, a recent study from the stratified Lake Lugano (Switzerland) showed 362 that the majority of the brGDGTs are produced in the lower, anoxic part of the water column rather than in the sediment 363 (Weber et al., 2018). Furthermore, the combination of brGDGT analysis with molecular biological methods revealed that 364 brGDGTs appeared to be produced by multiple groups of bacteria thriving under different redox regimes in this stratified 365 lake. Specifically, brGDGT-IIIa occurred in the entire water column and continuously increased with depth, whereas 366 brGDGT-IIIa' was mainly produced in the upper, oxygenated part of water column (Weber et al., 2018). Extrapolating the 367 ecological niches of brGDGT production in Lake Lugano to Loe Pool we can speculate that brGDGT-IIIa, which is 368 dominating the brGDGT signal in the Loe Pool sediments, is mostly produced in the lake during summer, when the 369 eutrophic state of the lake may seasonally cause the anoxic conditions favourable for its (i.e. brGDGT-IIIa) production. 370 However, our dataset does not allow to further pinpoint the time and depth of lacustrine brGDGT production, or whether 371 brGDGTs are solely produced in the water column of Loe Pool or also in the lake sediment. The C-normalized concentration of brGDGTs starts to increase around 23 cm, reaching a maximum concentration of 48.0 g 379 g -1 C at 11 cm depth (Fig. 6b). The increased brGDGT concentrations coincide with an increase in the degree of cyclisation 380 (Fig. 6c), which generally responds to a change in pH, where more cyclopentane moieties correspond to a higher pH 381 concentrations and DC in the sediments likely reflect the eutrophic conditions of the lake resulting from the increased 389 nutrient input to the lake (Coard et al., 1983). The DC has then recorded the increase in lake water pH associated with 390 eutrophication, whereas brGDGT concentrations express increased aquatic production. Due to remediation measures taken 391 by the local government in 1996 (~ 12 cm depth), the eutrophication has reduced over the past twenty years (Glendell et al., 392 2018). The partial recovery of the lake has likely resulted in a return to lower lake water pH, as manifested in the decrease in 393 the DC from ~ 10 cm depth upwards (Fig. 6c). 394 The process of eutrophication and subsequent recovery can also be recognized in the ratio between GDGT-0 and 395 crenarchaeol, which are isoprenoidal GDGTs produced by Archaea. Crenarchaeol is produced by ammonia oxidizing 396 Thaumarchaeota (Sinninghe Damsté et al., 2002) in aquatic environments (Schouten et al., 2000;Powers et al., 2004) and to 397 a lesser extent also in soils (Weijers et al., 2006a), whereas GDGT-0 is a membrane lipid that occurs in all major groups of 398 Archaea, but is indicative of methanogens and thus anaerobic conditions, with a typical ratio of GDGT-0 and crenarchaeol > 399 2 (Blaga et al., 2009). The ratio of GDGT-0/crenarchaeol in the sediments of Loe Pool is > 2 throughout the entire core, and 400 ranges between 10.9 and 24.3, indicating that at least the bottom waters of the lake have been (seasonally) anoxic over the 401 past 100 years (Fig. 6d), although the isoGDGTs may potentially be produced in deeper sediments. The ratio reaches its 402 maximum at 16 cm depth, suggesting that eutrophic conditions and bottom water anoxia were most severe around this time. 403 The recovery of the lake after the remediation measures is again reflected in the return to pre-1960 values at ~ 10 cm depth 404 (Fig. 6d). 405

Conclusions 406
In this study, brGDGTs were tested as a tracer for the transport of soil OC from different vegetation and land use types from 407 source (soil) to sink (lake Loe Pool) in the Carminowe Creek catchment with the aim to reconstruct the provenance of the 408 soil OC in lake Loe Pool sediments over time. Unfortunately, brGDGT signatures in the catchment soils are not distinct for 409 land use types, indicating that other environmental parameters have a larger influence on the distribution of brGDGTs in 410 these soils. Although temperature and precipitation can be considered equal for all soils due to the small size of the 411 catchment, changes in BIT index values and the relative contribution of 6-methyl brGDGTs along a part of the hilltop 412 transects indicate that soil water content (SWC) may exert a control on brGDGT signals, assuming that SWC increases 413 downslope. The regular rotation of cropland in this catchment and the relative long turnover time of brGDGTs in soils could 414 be another reason to explain the limited spatial variation in brGDGT signals. 415 Comparison of the soil-derived brGDGT signals to that of creek bed sediments reveals that the soil brGDGT signal is almost 416 completely overprinted by aquatically produced brGDGTs, indicated by a substantially higher fractional abundance of 6-417 methyl brGDGTs in the creek. Upon discharge into the lake, the creek brGDGT signal is replaced by and/or mixed with a 418 lacustrine in situ produced brGDGT signal, which is characterized by a relatively higher DC and lower MBT'5ME, as well as a 419 specifically high fractional abundance brGDGT-IIIa. Despite regular ploughing of the land, the absence of a profound rainy 420 season and limited relief likely limits the degree of soil mobilisation necessary to transfer the soil-derived brGDGT signal to 421 the lake sediments in the modern system. Still, downcore variations in GDGT distributions in the sediments of Loe Pool do 422 reflect local environmental conditions over the past 100 years. The degree of cyclisation of brGDGTs as well as the ratio of 423 isoprenoidal GDGT-0 and crenarchaeol produced by Archaea trace the historical record of lake eutrophication induced by 424 increased nutrient input from intensified agricultural activity in the catchment during the 1960s to 1980s, and its recovery 425 after measures taken by the owner since 1996. Our study shows that GDGTs in sedimentary archives are good recorders of 426 past environmental and land management (e.g. agricultural intensification, increased fertilizer use) change, although the 427 ability of brGDGTs to trace soil OC along a soil-aquatic continuum requires a higher degree of soil mobilisation. 428