Effects of a warmer climate and forest composition on soil carbon cycling, soil organic matter stability and stocks in a humid boreal region

ABSTRACT

soil C and N. Positive effects of warming both on fluxes to and from the soil as well as a potential saturation of stabilised SOC could explain these results which apply to the 40 context of this study: a cold and wet environment and a stable vegetation composition along the temperature gradient. While the entire study area is subject to a humid climate, a negative relationship was found between aridity and SOM stocks in the upper mineral soil layer for black spruce forests, suggesting that water balance is more critical than temperature to maintain SOM stocks. The use of climatic gradients to understand the impact of climate change on soil C 60 dynamics complements the information generated by soil warming experiments (Crowther et al. 2016;Rustad et al. 2001). If vegetation types and soil conditions are held constant, climatic gradients allow the observation of changes in soil fluxes with climate while avoiding short-term pulse effects that have been observed in manipulative warming experiments which may only be transient (Melillo et al. 2002). They can also be used to observe changes that takes long time to form, including the buildup of stable SOM (Lavallée et al. 2020).

Supprimé: climate
Supprimé: should also experience Climate gradient studies generally show shorter organic matter turnover times under 70 warmer climate (Tewksbury and Van Miegroet 2007;Ziegler et al. 2017). However, because both C fluxes to and from the soil are accelerated by an increase in temperature, the net effect on soil C accumulation varies when the effect on one flux is greater than on the other. For example, a Finnish thermocline study showed greater accumulation of soil C in warmer climate (Liski and Westman 1997) due to increased productivity, while the opposite was found for the SOM content of the soil organic layers of black spruce forests of Alaska (Kane et al. 2005;Kane and Vogel 2009) and for both organic and mineral soil layers in continental US (Garten 2012). Other studies have revealed stable SOC stocks along gradients of increased mean annual temperature in the Southern Appalachian region (Tewksbury and Van Miegroet 2007) and in Newfoundland and Labrador (Ziegler et al. 80 2017). In the latter study, increased fluxes to the soil at warmer sites were associated with higher C stocks in the organic layer.
A conceptual model of SOM dynamics, the Microbial Efficiency-Matrix Stabilization (MEMS) framework developed in Cotrufo et al. (2013) suggests that a larger inputs of labile plant material leads to larger and more stable SOM stocks. Thus, if warming enhances plant productivity, it could favor the accumulation of larger SOM reservoirs.
Stable SOM makes the bulk of SOM. For example, Andrieux et al. (2020) found that only 10% of total SOM could be considered as fast or bioreactive C. To the contrary, several studies that considered the decomposability of SOM have showed a higher content of bioreactive or poorly stabilized soil C in colder soils (Fissore et al. 2009;Ichikuza et al. 90 2007; Laganière et al. 2015;Norris et al. 2011) suggesting that cold temperature maintains reservoirs of easily decomposable SOM and consequently that cold soils may be more susceptible to C losses upon warming.
The interpretation of climate gradient studies is often complicated by changes in vegetation composition and in soil properties. The objective of the present study was to assess changes in SOC stocks, quality and C fluxes to and from the soil along a climatic gradient, while maintaining vegetation composition and soil properties as constant as possible for two important boreal forest types: (1) forests dominated by balsam fir (Abies balsamea (L.) Mill.), a fire-avoider that becomes abundant in landscapes with a long fire return interval, and (2) forest dominated by black spruce forests (Picea mariana (Mill.) B.S.P.), a fire adapted species that nevertheless can persist in the landscape in the absence of fire (Couillard et al. 2018). Wildland fires are projected to increase in number, size and intensity, due to climate-warming and fire frequency could increase by 1.5 to 4 times before 2100 in the Canadian boreal forest (Boulanger et al. 2014), a situation that would 110 likely lead to a reduction in the area covered by balsam fir forests.
We hypothesized that:  i) Stocks: Balsam fir sites, presumably because they produce greater amounts of litter of higher quality, as well as sites at the warmest end of the gradient should accumulate more stable SOM in line with the Microbial Efficiency-Matrix Stabilization (MEMS) framework developed in Cotrufo et al. (2013), which suggests that a larger inputs of labile plant material leads to larger and more stable SOM stocks. A larger stock of stable SOM should be apparent in total C stocks because it represents the largest share of total SOM (Andrieux et al. 2020).
 ii) Cycling: Warmer sites should exhibit enhanced C cycling to and from the soil.

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 iii) SOM bioreactivity: Warm sites should contain less bioreactive SOM because higher microbial activity at high soil temperature leads to its degradation (Laganière et al. 2015). The proportion of bioreactive SOM can be evaluated with the use of long-term incubation. A lower proportion of bioreactive SOM should also be reflected in a higher Q10 for soil respiration rate according to the "C qualitytemperature" (CQT) hypothesis (Laganière et al. 2015). Hence, balsam fir sites and warm sites should have greater soil C stocks and higher Q10.
In comparison to other transect studies dealing with SOC dynamics, this study considered two forest types. Forest composition can have major impacts on SOC storage and cycling 130 (Mayer et al. 2020). In addition, studying the impact of climate on SOC within vegetation type is important in the light of studies pointing out that vegetation shifts with climate change were found to be much more modest than expected. This has been related to non climatic factors including seed predation, pathogens and soil related limitations (Brown and Vellend 2014). In fact, land-use change and disturbances were found to have much greater effects on forest composition than climate change (Danneyrolles et al. 2019 Sites were at least a few km apart from each other and are the same sites as those described  Table 1 and 2 (FM, FR, LS, J2, TIR, PR, OEBS). On these sites, root trenching was conducted to distinguish the contribution of autotrophic from that of heterotrophic soil respiration and soil samples were collected for long-term laboratory incubations to determine the proportion of bioreactive soil C. Methods are described below. At each site, five 2 m x 2m sample plots where located on the perimeter of a 400 m 2 circular plot where forest tree mensuration measurements were conducted (see Larocque et al. 2014 Note: Temperature, precipitation and aridity were estimated daily over a 30-year period using BioSIM (Régnière, and Saint-Amant, 2008

Litterfall
Eight square littertraps (2862 cm 2 ) were installed on each site. Litterfall was collected at least twice a year during three years. Litterfall per trap was divided by the number of days 280 for the full sampling period and then multiplied by 365 to get annual estimates. Samples were kept at 3 o C,before being sorted to distinguish foliar material from other fine material (small branches, mosses, insects, insect frass, cones, flowers and unknown materials) dried at 70 o C. They were ground on a Wiley-mill and analysed for C and nitrogen (N) content as above.

Soil respiration in situ
In each sample plot, both in control and trenched plots, two 5 cm deep polyvinyl chloride (PVC) collars measuring 10 cm in diameter were inserted into the soil at a 1m distance from each other (10 collars per site) to measure total soil respiration (RS). On the seven intensive 290 sites, trenches were dug to a depth of approximately 30cm on a 2 x 1m area near each sample plot to sever roots. Trenches were lined with landscaping fabric and backfilled. Herbs and shrubs were removed from trenched plots periodically. Two PVC collars were installed in each plot to measure heterotrophic soil respiration (Rh). Installation was conducted in June and measurements started the following year to let the severed roots decompose and avoid a method-induced CO2 peak. CO2 efflux measurements were performed once a month from early May to mid-late-October. They were measured by placing the soil chamber (LI-6400-09) of a portable infrared gas analyzer (LI-6400, LI-COR Inc., Lincoln, NE) above the collar.
During soil CO2 efflux measurements, soil temperature at a depth of 10 to 15 cm was recorded as well as soil moisture (i.e. volumetric water content, % v/v) using a TDR-300 300 time domain reflectometry moisture probe (Spectrum Technologies Inc., Plainfield, IL). This relatively shallow depth was chosen because soil temperature at this depth fluctuates daily along the growing season. Soil temperature was measured continuously on the intensive sites with thermocouple wires attached to a Campbell Scientific data station, while WatchDog 100 Series Water Resistant Button Loggers were installed on the other sites. As detailed in Table 5, 5093 CO2 efflux soil collar measurements were performed on control plots and 2661 on trenched plot over the duration of the study. Measurements were performed over 4 years for most sites; three sites were sampled for only 3 years, and one site for two years at a rate of five to six measurement days per year (details in Table 5). Several parameters were derived from the respiration vs temperature relationship. The relationship of soil CO2 efflux 310 vs temperature was parametrized with a simple exponential equation (Buchmann 2000): Where: Rs is soil respiration in µmoles C m -2 * s -1 ; a and b are coefficients and Ts is soil temperature in o C at a depth of 10-15cm. [Eq.3]: Rs = RS10*2.7183 (0.0693*(Ts-10)) *3600*24*12*10 -5

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We compared the results from this calculation method with one using Q10 values that varies with site but that were the same for the whole season for the intensively studied sites. The overall difference between the two methods varied between sites and was of only 1% on average in yearly soil respiration (more details provided in SM2)

Partitioning into heterotrophic and autotrophic respiration
On the intensive sites, the same calculation as above was performed for the trenched plots to obtain an annual soil efflux per site for the trenched plot. The same parameters were fitted using Eq 1 to 3. The parameters derived from these equations were analysed and 370 discussed. However, because the values were higher than those generally reported in the literature (Rh averaged 77% of Rs) although close to the values reported by Lavigne et al. (2003) for cold sites, we suspected that the trenching was incomplete. We therefore used Where R (h or s) is respiration in g C m −2 year −1 . This value was subtracted from Rs to estimate Ra, autotrophic respiration.

Lab incubation
For the microcosm experiment, soil samples from four of the five plots per site were pooled by depth to yield composite samples for four intensively studied sites (Mild-Fir: FR; Warm-fir: LS; Cold-Spruce: J2 ; Warm-Spruce: PR; see Table 1 for site codes).
Organic and mineral layer samples, 6-and 40-g dry weight, respectively, were placed on a 390 layer of glass wool in 120 mL plastic containers (28 cm 2 surface area), wetted at field capacity and placed in 500 mL glass jars (Mason type). These microcosms were left to stabilize for one week at 2 o C after handling and were then incubated in the laboratory at 3, 10, 15 and 22°C at field capacity for 354 days. These temperatures were chosen to cover the range of soil temperatures that decomposers would experience in the field. Soil respiration rates were measured monthly during periods of 4 to 24h during which the lid was closed. The length of these periods depended on rates and CO2 evolution in order to keep concentrations within the calibration range of the IRGA, a LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA). The methodology used is described in Andrieux et al. (2020). At each measurement period, samples were flushed with distilled 400 water and NO3-N and NH4-N were analyzed by FIA (Quickchem 8500, Lachat Instruments, Loveland, Colorado). Flushing maintains the soil humid and prevents the accumulation of metabolic products that may interfere with the decomposition process.Using linear interpolation between measurement dates, the total amount of C and N mineralized during the full incubation was estimated and considered as bioreactive C or N.

Statistical analysis
The effects of DD, species and their interaction on the dependant variables (soil carbon pools and fluxes as well as soil respiration parameters (Rs10, Q10) and litterfall properties) were tested using proc GLM (SAS 2013 contained an average of 25% of estimated soil C stocks, while this proportion was 37% on spruce sites. Excluding the two sites where the 20-40 cm layer could not be sampled, the latter value falls to 33% and is thus close to that found for the balsam fir sites. No significant effects of tree species, DD and their interaction on soil C stocks were found in either OL or the top 0-20cm mineral soil layer (Table 3; Fig. 2a).
Supprimé: This variability could not be attributed to a single factor. The only site with particle size distribution classifying it as a sand, TIR, had a low C stock, but other sites that were not as sandy, also showed low C content (Tables 1 & 2).

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Supprimé: , as related to forest composition and cumulative annual degree-days (DD).

Supprimé:
Supprimé: for DD effects 3.2 Carbon fluxes to and from the soil Aboveground litterfall was linearly related to DD as well as to tree species, but the interaction of these two factors was not significant (Table 3; Fig. 2b, c), indicating that the slope of litterfall with degree-days did not differ between the two forest types. On average, fir forests produced 71% more foliar litter and 37% more total litter than black spruce ones.
The N concentration of foliar litter was higher for balsam fir than for black spruce and this 470 is reflected in lower C:N values for the former (Table 3; Fig.2d). This variable was remarkably stable for balsam fir across the temperature gradient, while it showed greater variability for black spruce but no trend with DD (Fig. 2d).
Cumulative soil respiration from May 1 st to October 31 st was linearly related to DD and was not significantly affected by tree species (Table 3, Fig. 3, SM1). Autotrophic, heterotrophic and total soil respiration rates were strongly and linearly related to degreedays (Fig.3). The slope of autotrophic respiration with DD was greater than that of heterotrophic respiration (SM3), although this difference was not significant at the 5% threshold (p=0.0832), suggesting that a higher share of autotrophic respiration to total soil 480 respiration for warm sites as observed by Lavigne et al. (2003). For two of the coldest sites, the values obtained from trenched plots (indicated by stars in Fig. 3) ,2 f: empty symbols). The Q10 values of trenched plots ranged from 1.55 to 3.12 and averaged 2.51 across sites and were not systematically lower nor higher than the values of their respective control plots (Table 3 Fig ,2 e).
Laboratory incubation indicated a higher proportion of bioreactive C and N in the organic layer than in the mineral soil (SM3). Incubation temperature had an overall significant effect for both soil layers and elements (Fig.4, SM5). The effects of DD and species on 530 bioreactive C and N were only significant in the OL (SM5). Warm and balsam fir sites showing consistently higher N and C mineralization rates (Fig. 4). This effect was significant at the 0.05 threshold for N but barely missed that mark (p=0.06) for species effect on C mineralisation (SM5). A warmer climate increases litterfall inputs as well as soil respiration rates without causing 560 a net effect on C stocks. However, we must be cautious with this interpretation because soil C stocks estimates have a high level of uncertainty, and it is not possible, given our sampling effort, to detect small changes (Yanai et al. 2003). Climate gradient studies in the boreal region have shown that warmer conditions can increase (Liski, & Westman, 1997), have no effect (Ziegler et al. 2017) or lower soil carbon stocks stocks (Norris et al. 2011, Kane & Vogel 2009) despite a higher productivity. These latter two studies were conducted in regions with a much drier climate than the one of this study and of that of the other studies cited above. This suggests that a negative effect of climate warming on SOM stocks appears above a certain threshold of aridity. When heat coincides with drought, which is more likely in drier climates, these events strongly reduce gross primary production (GPP) We tested the relationship between aridity and SOM stocks (SM6). No relationship was found for the organic layer. However, we found a negative relationship between aridity and SOM stocks in the upper mineral soil for all species combined (R 2 =0.29; p: 0.015) and a stronger relationship for black spruce sites (R 2 =0.44; p: 0.03), but no significant relationship for balsam fir (SM6). Because aridity is not related to temperature along our black spruce site gradient, these results indicate that aridity may have a more important role to play in maintaining boreal SOC stocks than temperature. The influence of these two 580 factors may often be confounded in climate gradient studies.
Under the cold and wet conditions at our sites, microbial decomposition as well as plant productivity are strongly temperature-limited and greater litter inputs due to warmer

Déplacé vers le haut [2]
: When heat coincides with drought, which is more likely in drier climates, these events strongly reduce gross primary production (GPP) but yield smaller reduction in ecosystem respiration, leading to strong reduction in net ecosystem productivity (NEP) (Von Buttlar et al. 2018). conditions are likely offset by a greater microbial decomposition. In addition, our study area does not appear to suffer from water deficit and tree growth is not sensitive to summer soil moisture (Girardin et al. 2021). Furthermore, the size of the SOM stock is not only 600 controlled by climate or net primary production (NPP) but is also influenced by soil types and drainage (Andrieux et al. 2018, Dalsgaard et al. 2016). While we tried to maintain these conditions as constant as possible in our site selection, they may still play an important control on soil C stocks overriding that of C fluxes (Dalsgaard et al. 2016). We were not able to explain the large variability in soil C stocks across sites. This property is highly variable at small scale and has notoriously been difficult to map (Paré et al. 2021).
A much larger dataset would be required to test for the effect of environmental factors on soil C storage and should include gradients of texture, drainage, vegetation composition and climate including aridity with as much as possible independence between driving variables. . Both species also showed a stable litter C:N ratio along the climate gradient, suggesting that the stoichiometry of C to N is not affected by climate.
The difference in litter C:N ratio between species is not surprising because black spruce typically show lower foliar N concentration than balsam fir (Paré et al. 2013). Despite this 620 difference for N, and perhaps greater N limitation in black spruce forests, the C litterfall rate of both species reacted positively and with the same slope to a warmer climate.
Litterfall has been found to be a good predictor of net primary production (Mahi et al. Supprimé: ¶ Our warmer sites generally had lower precipitation (Table 1).

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However, soil moisture measured during CO2 efflux measurements did not show consistent difference across the gradient (data not shown). larger dataset did observe a positive effect of warming on tree growth in Quebec for both 640 balsam fir and black spruce. They predicted that enhanced tree growth would be observed for an increase of 4 o C even without a change in precipitation for black spruce but would require a 15% increase in precipitation for balsam fir. Interestingly, we found a positive relationship between aridity and balsam fir litterfall and with total soil respiration for all species (SM6). Although a strong correlation between aridity and DD for balsam fir precludes disentangling the driving factors, the enhanced fluxes with warming suggests that water limitation is not an important driver of tree productivity and soil respiration in this wet climate.

Mis en forme : Surlignage
The linear trends that we observed between carbon fluxes and temperature using a 650 relatively small number of sites indicate that the assessment of biogeochemical fluxes, including soil respiration measurements or foliar litterfall may be more suited to study changes in NPP than the often-used tree ring analysis. These fluxes are less affected by stand maturity and are more directly connected to carbon capture by photosynthesis than stemwood biomass, which only represents from 10 to 30% of photosynthate allocation and depends on many physiological processes including allocation to below and aboveground parts, reproduction, defense and energy storage (Litton et al. 2007). Our results showed clear and linear trends with warming for litterfall and soil respiration, while tree ring analysis have shown divergent results in the Canadian boreal forest (e.g. Girardin et al. 2016vs Hember et al. 2019.

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Increased litterfall rate with a warmer climate was accompanied by a linear and positive response of soil respiration indicating a greater turnover rate of SOC as the climate warms.
These results are coherent with the observations of Ziegler et al. (2017) for a climatic gradient in balsam fir forests in Newfoundland and Labrador and suggest that soil C stocks can be maintained despite a greater turnover rate. These results are in line with an acceleration of C turnover rate observed at the global scale over the recent decades (Carvalhais et al. 2014). Interestingly, the maintenance of SOC pools despite a faster turnover, have also been observed for a tropical mountain climatic gradient (Giardina et al. 2014) suggesting that within a biome where water limitation to forest production is of  (2015) in that we did not find a lower reactivity to temperature, nor a greater amount of bioreactive OM in the soils of the cold sites. The increase in bioreactive organic matter on cold sites, observed in this latter study could be due to a greater abundance of bryophytes in the colder sites of this transect study, favouring the accumulation of bioreactive organic matter (Kohl et al. 2018 fractions of SOM are impacted by changes both in aridity and in temperature and to identify thresholds in aridity from which SOC stocks become vulnerable to warming.

CONCLUSION
Our results showed no evidence of net SOM losses or a reduction of the most active SOM fraction with a warmer climate and contrast with the conclusions that were reached in other studies derived from climate gradient studies or from direct soil warming experiments. Our results are applicable to the context of the study: a cold and wet climate where available moisture does not limit productivity or decomposition; closed-canopy coniferous forests that were not recently disturbed and the absence of change in forest composition along the The fact that we did not observed differences in total 760 SOM stocks in our study suggest that MAOM formation are not sensitive to climate perhaps because these reservoirs tend to saturate (Lavallée et al. 2020). Boreal regions show the highest concentrations of dissolved organic carbon (DOC) in the surface soil globally (Langeveld et al. 2020). This also suggest that the capacity for these soils to accumulate stable SOC is limited and that warming essentially changes the dynamics of unprotected POM. Cotrufo et al. (2021), POM dominates the dynamics of SOC cycling under cold and wet conditions. Apparently, warming both accelerated fluxes to and from the soil contribution to a stability of both SOM stocks and SOM 770 stability. ¶ Our results suggest that the dynamics of SOM that is not stabilised by association with minerals, that can be referred to as particulate organic matter (POM) has a large influence on C cycling in these soils. As indicated by Cotrufo et al. (2021), POM dominates the dynamics of SOC cycling under cold and wet conditions. Apparently, warming both accelerated fluxes to and from the soil contribution to a stability of both SOM stocks and SOM stability. ¶ ¶

Déplacé (insertion) [1]
Déplacé vers le haut [1]: Cotrufo et al. (2021), POM dominates 780 the dynamics of SOC cycling under cold and wet conditions. Apparently, warming both accelerated fluxes to and from the soil contribution to a stability of both SOM stocks and SOM stability.

Supprimé: environment
Supprimé: climate gradient. Other studies conducted under a wet climate corroborate these results.
Altogether, results from this study and from others suggest that a certain aridity threshold needs to be reached before a warmer climate has implications for SOM stocks.
Recognizing that the effects of climate change on SOM are not linear, identifying these thresholds would greatly improve our capacity to predict the fate of the important SOM 790 pool in boreal ecosystems under climate change. Our results also suggest that aridity may be more important than temperature in controlling SOM stocks.
Forest composition had effects on both SOM quality and the rate of C cycling. However, warming did not show interacting effects with forest composition. This suggest that differences in C cycling with forest composition remained stable with projected changes to temperature. These results indicate that the impact of climate change on SOM storage and dynamics needs to be studied both within and among forest ecosystem types to separate the direct effects of climate change from that of vegetation change.