We quantified demographic and functional trait responses in seedlings of six TDF tree species to increasing application rates of biochar and found contrasting impacts between the two trait types. While there were no effects on seedling survival and limited impacts on growth in two species under applications of 40 t/ha of biochar, seedling functional traits were more sensitive to the addition of biochar. Soil addition of biochar increased LCC, LAR, and SLA, thus indicating an improvement in the photosynthetic capacity of the seedlings (Markesteijn and Poorter 2009; Qian et al. 2021), while there were moderate increases in DMC of root, stem, and leaf material, possibly indicating an improvement in physiological tolerance to drought conditions (Hacke et al. 2001; Jacobsen et al. 2005). A large proportion of the variation in trait values was explained by inter-specific differences in trait responses (52%), while differences in intra-species trait responses explained on average 36% of the variation trait values. Despite this potential for adaptive phenotypic plasticity the experimental addition of biochar only accounted for a 11% of trait variability on average, with the notable exception of the LCC where up to 81% of its variation was due to biochar. Hence, biochar addition did not negatively affect the growth or the functional trait expression patterns of the seedlings of the six TDF species studied. Altogether, our findings suggest that there is a wide range of biochar addition schemes (e.g. from 5 up to 30 tons per ha) that either improve some parameters of most of the species, or at least do not negatively affect the performance of the most sensitive species. Hence, in our opinion biochar could be incorporated in large-scale tropical dry forest (TDF) restoration programs both at the seedling nursery level and at the field establishment stage without compromising seedling survival and growth, thereby potentially contributing to long-term C sequestration in the soil.
4.1. Overall Effects of Biochar on Tree Seedling Demographic and Functional Traits
We found positive main effects of biochar on key growth-related morphological traits (RDMC, SLA, LAR, RMF, LDMC, SDMC, DMC and RDMC) across species, this suggests that the addition of biochar as a soil amendment may improve the physiological tolerance to drought (Hacke et al. 2001; Jacobsen et al. 2005). The positive effects of biochar on the foliar traits (LCC, SLA and LAR) and root traits (RMF) may also improve the establishment of the seedlings in the field, as these traits are related to light capture, photosynthetic capacity, control of water losses through transpiration, and the capture and storage of water, nutrients, and seedling support, respectively (Markesteijn and Poorter 2009). The addition of biochar has a strong impact on soil nitrogen dynamics, increasing its adsorption and mitigating its leaching losses (Clough et al. 2013). This potential increase in nutrient availability may help to explain the higher capacity of the plants to synthesize and accumulate more chlorophyll in their leaves.
However, we did not find main effects of biochar on seedling survival, TDM, LDM, RDM, LMF, Stem, Height, or SRL, and moderately negative effects on SDM and SMF. This is in contrast to the prevailing pattern of mostly positive plant growth responses to biochar additions. This general increase of growth has been found on various crops (Biederman and Harpole 2013; Liu et al. 2013), on woody plants (Thomas and Gale 2015), and also on pioneer herbaceous species (Gale et al. 2017). Despite that, prior studies have also found neutral or negative responses (e.g., Spokas et al. 2012; Gale & Thomas 2019; Gonzalez Sarango et al. 2021). The reasons for such disparities can be the great variability of biochars and soil properties, the range of rates applied, and also the specific responses of the plant species assessed due to their different ecological strategies. In our study, the response to biochar was dose-dependent, only arising a slightly negative growth after adding 40 t/ha and only in two out of the six species tested. This observation is in line with previous works reporting moderately negative effects of biochar on aboveground plant biomass production from 40 t/ha onwards (Gale and Thomas 2019). The fact that this negative effect only affected two of the species tested, however, points out to the highly variable and species-specific plant responses to biochar. With all, these results highlight the wide margin (e.g. from 5 up to 30 tons per ha) for a safe application of biochar in TDF restoration programs before producing negative effects on tree seedlings growth even on the most sensitive species.
4.2. Species Responses to Biochar
Our data show inter-specific variation in trait responses to addition of biochar, where there was greater allocation to aboveground biomass (high TDM, SDM, SLA) in G. ulmifolia, while in C. alata there were reductions in RMF, SMF, and increases in LMF and SLA. The allocation of biomass to plant organs varies with species, ontogeny, and environmental conditions (Poorter and Nagel 2000). Dry forest species, such as C. alata, limit water losses through reductions in amount of transpiration tissues (lower Leaf_area, SLA, LAR) and improved access to water in deeper soil layers (higher RMF) (Poorter and Markesteijn 2008), whereas fast-growing species, such as G. ulmifolia, are characterized by acquisitive foliar traits and greater allocation of biomass to aboveground structures under high levels of nutrient availability and greater allocation of biomass to belowground structures under nutrient limitation (Lanuza et al. 2020). Our data show that seedlings of TDF species modulate biomass allocation depending on the biochar rate applied, and this suggests that they did so in response to shifts in resource availability as has been observed in previous studies (Lanuza et al. 2020).
Our results indicate that greater variation in above- and below-ground trait responses to biochar was due to inter-specific, rather than intra-specific (ITV) differences and we found that addition of biochar increased the plasticity index of above- and below-ground functional traits (SRL, SDM, RDM, LDM, LCC). We found that ITV of our experiment was slightly higher than that reported by Poorter et al. (2018); Siefert et al. (2015), and similar to levels for three dry forest species subjected to contrasting levels of nutrients, irrigation, and herbivory (Lanuza et al. 2020), indicating these species may show high level of adaptability to shifts in environmental conditions (Poorter et al. 2018).
We found that addition of biochar accounted for an average of 11% variation in trait responses across species, yet explained 81% of species variation in LCC, while the overall proportion of variation in LCC explained by biochar was 1.3%, indicating the sensitivity of this functional trait to water stress, given drought affects photosynthesis (LI et al. 2006). Biochar improves water retention capacity due to its internal porosity and by increasing the interpore volume of soils (Liao and Thomas 2019), and applications of biochar have been shown to improve water use efficiency of pioneer herbaceous seedlings by 44% (Gale et al. 2017), but also reduce leaf N content and LCC in tomato seedlings (Akhtar et al. 2014).
The proportions of inter-specific and intra-specific variation in trait responses to biochar addition ranged between 9.5 and 8.7–78%, respectively. Traits related to tissue quality and toughness (DMC of root, stem, leaf) are expected to express low levels of ITV, as they tend to be phylogenetically conservative (Chave et al. 2006); this was evident in our study for SDMC and RDMC, but was higher (61%) for LDMC. We found a low ITV for SLA, supporting findings reported by Poorter et al. (2018), but in contrast to controlled studies that show marked responses in leaf traits to shifts in ambient light levels, to enhance light capture (Poorter et al. 2009; Sterck et al. 2013).
Our data show high levels LMF, SRL, and LAR in T. rosea, C. odorata, and S. humilis, that are coupled with low levels of DMC of leaf, stem, and roots, being the latter traits typically correlated with the physiological tolerance to drought (Hacke et al. 2001; Jacobsen et al. 2005). Thus, it is likely that these species may be susceptible to water deficit, in contrast to G. ulmifolia that was characterized by high levels of DMC, height and robustness (H:D ratio), indicating adaptations for light capture, water transport, support, and tolerance to wind damage and drought conditions (Poorter 1999; Haase 2008). We found that above-ground (LMF) and below-ground (RMF) trait responses to biochar mirrored relative allocations of biomass, as reported by Lanuza et al. (2020) for dry forest seedlings subjected to contrasting levels of fertilization and that are similar to responses to drought conditions, where species tend to reduce biomass allocation of LAR and LMF and increase allocation to RMF (Poorter and Markesteijn 2008; Markesteijn and Poorter 2009). Competition for above-ground and below-ground resources tends to be dynamic during the seedling stage, when acquisition of sufficient water, nutrients, and light is essential for sustained growth (McMurtrie et al. 2008; Poorter et al. 2012; Ågren et al. 2012; Fatichi et al. 2014).
Previous research has shown limited effects of RMF and LMF on below- and above-ground resource foraging, respectively (Poorter and Nagel 2000). Our study showed that limited investment in root biomass (low RMF), as found for C. odorata, S. humilis and T. rosea, may be offset by cost-effective root growth, as indicated by large root length per unit of biomass invested (high SRL), whereas low biomass investment in leaf material (low LMF), as found for G. ulmifolia, may be offset by large leaf area per unit of leaf biomass invested (high SLA). This compensation strategy in above- and below-ground biomass allocation has been demonstrated in response to drought conditions (Markesteijn and Poorter 2009).