Introduction

Soils are a major reservoir of terrestrial carbon (C), storing more C (2 344 Gt C, up to 3 m depth) than terrestrial biomass (560 Gt C) and atmospheric pools (750 Gt C)1,2,3. A small change in soil organic C (SOC) may have a great impact on atmospheric C and subsequently on the earth’s climate4. Land-cover change is one of the main factors that can alter SOC content5,6. One of the major sources of land-cover change, especially in tropical and subtropical regions, is encroachment by native woody plant species. In South Africa, this problem is particularly acute; encroachment affects about 10–20 million ha, seriously reducing the productivity for a country where >70% of its agricultural area is largely grazing lands7.

Jackson et al.8 examined the effects of vegetation change on SOC at six paired grassland and invaded woody sites along a precipitation gradient in the south-western USA, testing relationships between biomass C gains and SOC losses for common native woody invaders, such as Prosopis (mesquite), Larrea (creosote) and Juniperus (juniper) species. In that study, they found that there was a greater increase in SOC stored in the encroached sites than in the open grasslands in drier locations (i.e., below ~400 mm mean annual precipitation (MAP)). However, Jackson et al.8 also observed lower values of SOC in encroached sites than in open grasslands in the more humid locations. Other data show that generally organic C and total N pools in soils beneath shrub canopies increase linearly with time since shrub establishment9. Findings from several studies10,11 indicated that woody plant encroachment is a potentially, but highly uncertain, carbon sink. A subsequent meta-analysis of SOC data on woody plant encroachment found that the change in soil organic C in response to woody plant encroachment was highly variable and unrelated to MAP12.

Soil bulk density and clay content mediate the magnitude and direction of change in SOC with woody encroachment. In encroached grasslands SOC decreased as soil bulk density increased12 while SOC accumulated with increasing clay content13,14. Barger et al.12 reported that SOC accumulated linearly with increasing clay content (r2 = 0.76), presumably reflecting greater occlusion and protection of organic matter afforded by clay micelles13,14. They also found that soil C accumulation decreased as soil bulk density increased, with C gains (r2 = 0.52) being confined to soil densities less than 1.6 g cm−3.

Woody plant encroachment in tropical grasslands usually alters the ecosystem from C4 plants (African grasses use this photosynthetic pathway) to C3-dominated woody vegetation13,14. The change in the dominant photosynthetic pathway offers a unique opportunity to use the natural abundance of 13C to evaluate changes in C cycles due to woody plant encroachment of grasslands. Variations in 13C/12C ratios of plants utilising the C3 and C4 photosynthetic pathways provides a natural tracer when a C4 community type (δ13C ≈ −14‰) is replaced by a C3 community (δ13C ≈ −27‰) or vice versa15,16, allowing for the differentiation of the C derived from C4 vegetation from C derived from C38,17,18.

SOC in encroached grasslands can also be enhanced by an increase in available soil nitrogen. Change in nitrogen availability in ecosystems is likely to drive net primary productivity and thus C sequestration19. The frequent increase of N2-fixing woody species in grasslands20,21 has strong potential for altering the N-cycle, primary production, and other key ecosystem processes21,22. Plant species are likely to influence ecosystem N cycling via alterations in N-use efficiency and by changing N inputs and losses23. Changes to the soil total N pool via the shift in the balance of N inputs versus losses should be apparent in the natural abundance of 15N in the plant-soil system21.

In this study, we quantified and compared C and N pools (measured as concentrations and mass stocks) between adjacent woody- encroached and open-grassland plots along a precipitation gradient (300 mm to 1500 mm per annum) in South Africa. The major encroaching species in our sites (except at the 300 mm MAP site in Middelburg) were potentially N2-fixing shrubs (Vachellia karroo, Senegalia mellifera, V. tortilis and V. erioloba). We focused on responses in C sequestration, and in particular its relationship with MAP, as a key continental scale driver of ecosystem function24. We recorded SOC, N, δ13C and δ15N at depth intervals of 0–10, 10–30, 30–60 and 60–100 cm and determined the δ13C and δ15N isotopic ratio in plant leaves and plant litter. We also determined the contribution of C4 plants to SOC in the encroached grasslands. Our study sites contained mostly warm-season C4 grasses (Table 1). The sites in decreasing order of precipitation were KwaMbonambi (1500 mm yr−1), Stanger (900 mm yr−1), Bergville (750 mm yr−1), Bloemfontein (500 mm yr−1), Pniel (350 mm yr−1) and Middelburg (300 mm yr−1). At each site, we examined six encroached and six open grasslands. We predicted that in encroached grasslands there would be a negative correlation between changes in SOC and MAP8. We also predicted a negative correlation between C change due to woody plant encroachment and soil bulk density and a positive correlation between C change due to woody encroachment and silt and clay content10. We predicted similar relationships for soil N because of the tight relationship between soil C and N8. We also predicted that δ13C will become less enriched in shrub-encroached grasslands compared to open grasslands because of transition from C4 grasses to C3 woody-shrubs and trees15,16.

Table 1 Study site descriptions.
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At the most humid site (1500 mm MAP), in the 0–10 cm layer, SOC concentration and SOC stocks were significantly greater (F1, 10 = 83.70, P < 0.0001 and F1, 10 = 103.9, P < 0.0001) in the open grasslands than in shrub-encroached grasslands (Fig. 1). This humid site lost 50.5% of SOC stocks in the 0–10 cm soil layer (Table 2). The loss in SOC stocks to 100 cm depth due to woody plant encroachment at this humid site was almost 50%. At the 900, 750 and 500 mm MAP sites there were generally no differences in SOC concentrations and stocks between encroached and open grasslands in the uppermost soil layer (Fig. 1, Table 2). However, the 750 and 500 mm MAP sites gained carbon stocks slightly at 0–1 m depth, ranging between 9.2% and 25%. In the semi-arid sites of 350 and 300 mm MAP, SOC concentration in the 0–10 cm soil layer was higher (F1,10 = 5.06, P = 0.048 and F1,10 = 16.73, P = 0.002, respectively) in encroached grassland than in open grasslands (Fig. 1). The 350 and 300 mm MAP sites had higher SOC stocks (F1, 10 = 11.26, P < 0.007 and F1, 10 = 11.26, P < 0.022) in the encroached grasslands compared to open grasslands in the upper soil layer.

Figure 1
figure 1

Soil organic carbon concentrations (g C kg−1 soil) vs. depth (cm) along a precipitation gradient (300–1500 mm MAP) in encroached grasslands (black circles) and open grasslands (open circles). Error bars represent standard errors. Some encroached and open grassland data points at the 350 and 300 mm sites completely overlap. Means within the same depth different letters are significantly different (Tukey post hoc test, p < 0.05).

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Table 2 Soil organic C and N stocks (Mg/ha) to 1 m depth and % change in C and N after shrub encroachment.
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There was a significant negative correlation between change in SOC stocks in open versus encroached grasslands in the 0–100 cm soil layer and mean annual precipitation (MAP) (r2 = 0.83, P = 0.012 (Fig. 2a)). In contrast, there was no significant relationship between the change in SOC stocks (between open and encroached grasslands) and soil bulk density (r2 = 0.05, P = 0.68 (Fig. 2b)) and between change SOC stocks and silt and clay content (r2 = 0.02, P = 0.77 (Fig. 2c)).

Figure 2
figure 2

Relationship between changes in SOC stocks (%) at 0–100 cm depth with woody encroachment in pairs of encroached and open grasslands and (a) mean annual precipitation (MAP) in mm; (b) silt and clay content as percentage; (c) bulk density in g cm−3. A positive value is achieved because there is more SOC in encroached soils than in open grassland soils and vice versa.

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Nitrogen was significantly higher in shrub-encroached grasslands than in the open grasslands in the 0–10 cm soil layer in the 900, 500, 350 and 300 mm MAP sites (Fig. 3). The correlation between the change in total N stocks of encroached versus open grasslands in the 0–100 cm soil layer and MAP tended to be negative and marginally significant (r2 = 0.62, P = 0.064; data not shown). There was no significant relationship between total N change between encroached and open grasslands and soil bulk density (r2 = 0.001, P = 0.89) or between total N change and silt and clay content (r2 = 0.026, P = 0.76).

Figure 3
figure 3

Soil total-N concentrations (g N kg−1 soil) with depth (cm) along a precipitation gradient (300–1500 mm MAP) in encroached grassland (black circles) and open grasslands (open circles). Means at the same depth with different letters are significantly different (Tukey post hoc test, p < 0.05).

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In the encroached grasslands, δ13C was generally more negative in the plant foliage and plant litter compared to the top soil. The open grassland showed the opposite trend, with the plant material being more enriched in δ13C than the top soil (Fig. 4). δ15N was generally more enriched in the top soil compared to the plant material in both the encroached and open grasslands (Fig. 4).

Figure 4
figure 4

δ13C (‰) and δ15N (‰) in open grasslands (open circles) and encroached grasslands (black circles) in plant material (a,d), plant litter (b,e) and top soil (c,f).

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At 0–10 cm depth, the δ13C values were significantly different between encroached and open grasslands in the 1500, 900, 500 and 350 mm MAP sites while in the 300 and 750 mm MAP sites there was no significant difference in δ13C between the two encroached and open grasslands (Fig. 5). There was generally δ13C enrichment with increasing depth across the precipitation gradient. There was no significant correlation between the change in δ13C between encroached and open grasslands and mean annual precipitation (MAP) (r2 = 0.03, P = 0.35). There was no significant enrichment of δ15N with increasing depth in both the encroached and open grasslands (Fig. 6) across all sites. Encroached grasslands had a significantly higher δ15N than open grasslands in the 500 MAP site (F1, 10 = 8.72, P = 0.01) in the 0–10 cm soil layer (Fig. 6). δ15N change between encroached and open grasslands was also not significantly correlated with MAP (r2 = 0.20, P = 0.46). Although there was a positive relationship between δ15N change and silt and clay content, the relationship was marginally significant (r2 = 0.64, P = 0.06). However, we found a significant and negative correlation between δ15N change and soil bulk density (r2 = −0.86, P = 0.008).

Figure 5
figure 5

δ13C (‰) values in encroached grassland (black circles) and open grasslands (open circles) with depth (cm) in South Africa. Error bars are standard errors of the mean. The reference standard was Pee Dee Belemnite. Means at the same depth with different letters are significantly different (Tukey post hoc test, p < 0.05).

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Figure 6
figure 6

δ15N (‰) values in encroached grassland (black circles) and open grasslands (open circles) with depth (cm) in South Africa. Error bars are standard errors of the mean. The reference standard was atmospheric nitrogen. Means at the same depth with the same letter are not significantly different (Tukey post hoc test, p < 0.05).

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Assuming that there is no isotopic fractionation during decomposition25,26, the isotopic-mixing model indicated that the percentage contribution of C3 plants (i.e. woody species) to SOC in the encroached grassland increased from 24.7% in the 0–10 cm soil layer to 31.6% in the 60–100 cm soil layer at the 1500 mm MAP site. From the 900 to the 300 mm MAP sites, the contribution of C3 plants to SOC generally decreased with increasing depth in the encroached grasslands (Table 3). The C3 contribution to SOC ranged from 64.6 to 24.7% in the uppermost soil layer and between 8.59 to 35.7% in the 60–100 cm soil layer. The lowest C3 contribution was at the 750 mm MAP site which ranged from 3.6 to 17.6%. We acknowledge that the isotopic-mixing model used here does not take into account the fractionation of δ13C with depth due to decomposition27.

Table 3 Carbon derived from encroaching C3 woody plants in encroached sites.
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Our study lacked a direct test of SOC change independent of other covarying factors, such as fire, grazing intensity and microbial activities. Nonetheless, our findings agree with several studies8,28,29 that found a negative correlation between change in SOC and MAP due to shrub encroachment. Our results, however, were contrary to a review by Barger et al.11 who showed that C response to shrub encroachment was highly variable and unrelated to MAP. Additionally, our findings also show that shrub encroachment sequesters more C at MAP < 900 mm while the study by Jackson and colleagues8 found a threshold value of approximately 400 mm MAP. The variance in the threshold MAP might be due to large differences in SOC level between their studies and our study sites, with their most arid site (230 mm MAP) having more C than our 500 mm MAP site.

The increase of SOC in the drier sites due to shrub encroachment is in accordance with the findings from several studies8,30,31. This increase in SOC agrees with the well-described island of fertility14,28,32. Shrubs and trees develop extensive root systems that extract nutrients from the horizontal and vertical planes30, leading to local nutrient recycling and greater biomass production associated with organic matter accumulation29,30. The accumulation of SOC in the drier shrub-encroached grasslands can also be due to greater rates of primary productivity in the more arid sites because the encroached grasslands had greater biomass than open grasslands. Greater primary productivity increases soil organic matter input in the soil beneath deep-rooted shrubs through increased surface litter and accumulation of complex organic compounds, which are resistant to decomposition14,24. The humid site (MAP = 1500 mm) had more C in the open grassland compared to the encroached grassland. This finding for our most humid site was contrary to that of Chiti et al. (2016)33 who found that woody plant encroachment resulted in the increase in SOC. However, our results were consistent with the findings of Jackson et al.8. In high-precipitation areas, grasslands are highly productive and allocate a large proportion of SOC belowground with greater root turnover rates8.

Based on the carbon isotopes, this study also indicated that the current organic matter inputs are not in isotopic equilibrium with soil organic matter in shrub-encroached grasslands. The δ13C values of the plant foliage was generally lower than those of soil organic carbon across all sites. In the shrub-encroached grasslands, δ13C values of organic inputs were characteristic of C3 plants (−32 to −28‰) while δ13C values of the associated SOC was −25 to −14‰. These contrasting values suggest that our shrub-encroached grasslands dominated by C3 plants were once occupied by C4 grasses.

δ13C soil values in our open grasslands (−18 to −12‰) where generally comparable to other C4 grasslands and savannas across the world (−16 ± 2.2‰)34. Thus, the δ13C values across all our six study sites showed that SOC in open grasslands came from a C4 dominated ecosystem. The discrepancy between δ13C values of vegetation and soils in open grasslands of our drier study sites may reflect lateral intrusion of C3 plant roots from the shrub-encroached grasslands. Shrubs in semi-arid region have been shown to have extensive roots that can extend to several meters35.

In our drier sites (500, 350 and 300 mm MAP sites), a strong memory of C4 plants that once dominated these sites was shown by SOC δ13C values at depths deeper than 30 cm, while the humid sites (1500 and 900 mm MAP) showed a significant δ13C change across the whole range of depth studied. This may indicate a quick SOC turnover in humid sites compared to semi-arid sites. It may also suggest that shrub-encroachment is more recent in semi-arid sites relative to the humid sites. Recent changes in C4 to C3 productivity might be evident in the δ13C of SOC near the surface were SOC turnover is most rapid and current organic input are concentrated.

A particularly strong memory of C4 plants that occupied the shrub-encroached grassland was very evident in δ13C values for our 750 mm MAP sites, which ranged from −14 to −12‰ across the soil depth. This finding indicates that organic carbon at this shrub-encroached site was exclusively from C4 plants and that little SOC was derived from the present C3 vegetation. This very strong C4 memory can be attributed to the high clay content of the soil at the site34. The finding is consistent with other studies which showed slow turnover of clay associated organic carbon15,34. Organic carbon associated with clay soils contains aliphatic hydrocarbons which are inert from carbon turnover36.

In the 1500 mm MAP site the δ13C signature in encroached grasslands indicated that new SOC from woody plants has been incorporated into the soil. However, the finding that this humid site had low SOC may indicate that the rate at which SOC is being lost is higher than that being added by the C3 plants. The woody plants have added approximately 24.7% of SOC compared to 50.5% lost due to encroachment. This humid site had also the lowest SOC and SN compared to our other humid sites. The isotopic mixing model also shows that C3 SOC contribution for this site increased with depth from 24.3% in the 0–10 cm depth to 31.6% in the 60–100 cm depth. These findings may indicate high nutrient leaching accelerated by the sandy soil type and high MAP.

Although the encroached grasslands are dominated by C3 plants, the bulk of the SOC likely comes from the C4 plants according to the mixed-isotopic model we used. The higher SOC δ13C deeper in the soil may be interpreted as supporting this conclusion, although this depends on there being limited discrimination against 13C during respiration/decomposition25,26. We have shown elsewhere, using aerial and fixed-point photography, that several of the sites (300 mm, 350 mm and 750 mm MAP) were open grasslands 20 to 50 years ago31,32,33,34. Although 1500 mm MAP site possibly indicated a decrease in the contribution of the C4 plants in SOC with increasing depth, the bulk of the SOC is still potentially derived from the C4 plants. This decrease could also mean that the 1500 mm MAP site has been encroached for a much longer period than in the other sites.

Soil N was higher in the encroached grasslands compared to open grasslands at the 900, 500, 350 and 300 mm MAP sites over the 0–10 cm soil depth probably because most of the encroaching woody species at these sites are leguminous N-fixing plants such as Vachellia erioloba, Vachellia karroo and Senegalia mellifera. N2-fixing plants ameliorate and increase N in the soil35. However, at the 1500 mm MAP site, soil nitrogen was significantly higher in the open compared to the encroached grasslands, which is consistent with the SOC trends at this site. This finding is consistent with results from other African savanna ecosystems where sites encroached by woody species have been compared with open grasslands37,38,39.

There was a general decrease of plant material δ15N values with MAP. This finding is consistent with many other studies that showed a negative correlation between δ15N and MAP40,41,42. A plausible explanation for this decrease in δ15N with increasing MAP is that photosynthetic activity become more water-than nutrient- limited41. Arid environments have high nutrient availability therefore have a more open N cycle resulting in a less negative δ15N compared to humid ecosystems.

There were no significant differences in δ15N values between shrub-encroached and open grasslands across all depths as would be expected if the source of the increase in soil N pools was N fixation21,37. This might suggest other processes affecting 15N concentration, such as N pumping from deeper soil layers and isotope fractionation during litter decomposition21,43 and denitrification39, to be contributing to soil nitrogen levels in the shrub-encroached grasslands.

This study generally showed shrub encroachment may result in SOC gains in areas with a threshold value between 750 mm and 900 mm MAP. Most of the SOC in the shrub-encroached grasslands came from the C4 plants that previously occupied the area. The significant difference between our study and the North American study of Jackson et al.8 which showed a threshold of 400 mm indicates that extrapolation across continents is problematic, as has been indicated for savannas elsewhere44.

Methods

Soil samples were collected between October 2013 and March 2014 at six different sites. The site are described in Table 1 according to Mucina and Rutherford (2010)45. Each site was divided into encroached and unencroached sections, using adjacent sections to minimize differences in topography. In three of our sites (300, 350 and 750 mm MAP) there is documented evidence46,47,48,49, while for the other three sites we have strong oral evidence, suggesting that our sites were once open grasslands 20–50 years ago. Encroached sites were defined as those areas with >40% shrub cover. Six 1 m-deep and 1 m-wide pits were dug at random at each site in each section. Within each pit, two soil cores of 100 cm3 each were sampled (drilled horizontally into the side of the pit) at centre of each of the four different depth intervals (0–10, 10–30, 30–60 and 60–100 cm). One core was used to quantify soil bulk density (BD) and soil texture, and the other core was used to determine soil organic carbon (SOC), total nitrogen (TN), δ13C and δ15N values. Bulk density and soil texture were quantified using the core method and pipette method, respectively50,51. The soil cores designated for SOC, TN, δ13C and δ15N were air-dried and passed through a 2 mm sieve. Soil samples for SOC and δ13C were treated to remove soil carbonates. To remove soil carbonates, soil samples were treated with 1N H2SO4/5% FeSO4 and double-checked for complete inorganic C removal51. Soil samples were air-dried at 40 °C and then sieved (2-mm mesh). Generally, our soil samples were free of rocks. Total SOC and N were measured using a Europar elemental analyser (Germany) at BemLabs (Somerset West, Western Cape). The SOC and N stocks were calculated using the following equation:

$${S}_{s}={x}_{1}{x}_{2}{x}_{3}$$
(1)

where Ss is the SOC or nitrogen stocks (Mg C ha−1); x1 is the C or N concentration in the soil material (%); x2 is the soil bulk density (Mg m−3); x3 is the thickness of the soil layer (cm).

We collected plant foliage from the five most abundant species at each site in each landcover area (encroached and open grassland). We sampled litter layer composed of plant debris in different stages of decomposition using a 15 cm × 15 cm wooden frame around each station. Both the plant material and the litter layer were air dried for 3–4 days and then oven dried at 60 °C. The plant foliage and the litter-layer material were then ground before the determination of their δ13C and δ15N signatures.

For δ13C and δ15N values, c. 40 mg of soil, 2 mg of plant foliage and 5 mg of litter- layer material was weighed into tin capsules (Elemental Microanalysis Ltd., Devon, UK) and combusted in a Thermo Flash EA 1112 series elemental analyser; the gases were fed into a Delta Plus XP isotope ratio mass spectrometer (Thermo Electron Corporation, Milan, Italy). The C isotopic ratio of a sample were expressed relative to the Pee Dee Belemnite standard52 and the N isotopic ratios was expressed relative to atmospheric nitrogen53.

Assuming complete and unbiased mixing in the soil, we used the δ13C values of soils (Fig. 5) and our mean δ13C values for the plant material of C3 (shrub) and C4 (grass) species (Fig. 4) present at our sites to estimate the relative proportion (%) of soil organic matter derived from C4 and C3 photosynthetic pathway sources with an isotopic-mixing model8:

$${{\rm{FC}}}_{3}=({{\rm{\delta }}}^{13}{{\rm{C}}}_{{\rm{soil}}}-{{\rm{\delta }}}^{13}{{\rm{C}}}_{{\rm{C4}}})/({{\rm{\delta }}}^{13}{{\rm{C}}}_{{\rm{C3}}}-{{\rm{\delta }}}^{13}{{\rm{C}}}_{{\rm{C}}4})$$
(2)

where FC3 is the carbon fraction derived from C3 sources, δ13Csoil is the measured δ13C of the soil sample, δ13CC4 is the mean δ13C (‰) of C4 sources, and δ13CC3 is the mean δ13C (‰) of C3 sources37. The isotopic mixing model used here does not consider δ13C fractionation with depth due to decomposition. Several studies have shown no indication of isotopic fractionation during decomposition25,26, while several other studies have shown various 13C fractionations due to decomposition27.

The data on SOC and N concentration, and SOC stocks, δ15N and δ13C values attributable to the effects of shrub encroachment and soil depth were analysed using a multivariate ANOVA analysis (MANOVA) to reduce problems with Type I error because we examined several dependent variables simultaneously. The datasets used in this study are available from the corresponding author on request.