1 Introduction

Biochar is a carbon-rich material produced by biomass pyrolysis (wood, crop residues, and manure) under anaerobic conditions (Ok et al. 2015). Their high porosity, large surface area, and strong persistence are promising for promoting microbial abundance, immobilising or detoxifying various pollutants, and buffering greenhouse gas emissions (He et al. 2021). Therefore, biochar use has been recommended in various situations and is generally considered beneficial to ecological systems. For example, biochar high porosity and large surface area can improve microorganism ecological activity by providing shelter and nutrients (Gorovtsov et al. 2020). In addition, previous studies have suggested that biochar can alter the living environment, and thus soil animal activity. For example, biochar can increase soil porosity and structure (Jouquet et al. 2006), creating active channels for larger soil animals (such as earthworms and termites). A lower soil bulk density is associated with an increased number of large soil animals (McCormack et al. 2013).

Soil animals are sensitive to environmental changes such as temperature, moisture, pH, and nutrient availability, leading to significant changes in their abundance, body weight, and species composition (Liu et al. 2020). For example, biochar application could inhibit body weight gain and behaviour and even induce neurotoxicity in Caenorhabditis elegans Maupas due to the environmentally persistent radicals in biochar (Lieke et al. 2018). In contrast, Gruss et al. (2019) found that biochar addition improved soil chemical properties (particularly organic carbon content and cation exchange capacity), which significantly increased the mean number of soil mesofauna (mites and springtails) and improved soil biological quality (Gruss et al. 2019). Moreover, biochar addition to acidic soils increases soil pH and microbial earthworm and ant activity, suggesting suitable ecological conditions (Van Zwieten et al. 2009). These few examples illustrated that there was not yet a consistent trend regarding biochar addition effects on soil fauna and their activity and that more in-depth studies, including further species, are needed to clarify the underlying mechanisms.

Ants are excellent model organisms for tracking changes in habitat because of their ecological success, diversity, and ubiquity (Elizalde et al. 2020; Salas-Lopez et al. 2018). As indicator species, ants can complement or even replace nematodes and earthworm functions in dry and hot habitats (Andersen 1991). Ants typically constitute 15–20% of the total animal biomass in terrestrial ecosystems, and play a fundamental ecological role in soil development, energy cycling, insect populations, and plants growth, and provide numerous ecosystem services such as seed dispersal, soil aeration, and nutrient cycling (Schultheiss et al. 2022). Previous studies have suggested that biochar application increased Tetramorium caespitum L. ant numbers in a vineyard (Castracani et al. 2015), but repelled the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Chen et al. 2022). The diversity and species richness of ant communities could be altered because of the increased soil temperature (deepen soil color but reduce albedo and soil thermal conductivity) and water content, and balanced pH after biochar application (Castracani et al. 2015). Therefore, previous studies focusing on community diversity and soil animal toxicology have not reached a consistent conclusion. Other ant traits have also been proven effective in assessing the ecological and environmental impact of human activity. For example, ant morphological traits are associated with changes in species affinity for different macrohabitats under human-induced land-use changes, as well as food and niche preferences (Salas-Lopez et al. 2018), Moreover, morphological characteristics are effective behavioural thermoregulators and, thus, are adaptive and sensitive to a wide range of temperatures. For example, Weber’s length (WL), which measures the alitrunk length, is often used as a surrogate for total body length (TL) and is indicative of insect metabolism, thermal tolerance, foraging duration, and respiration rate (Peters et al. 2016). A community functional diversity measure through time has emerged as a key concept to explain ecosystem resilience to environmental change, to describe ecosystem processes, and to evaluate ecosystem services, at least in part (Drager et al. 2023; Weiss and Ray 2019). Therefore, studying biochar application effects on ant morphological characteristics can provide essential information for a systematic understanding of the ecological role of biochar.

In general, invasive species, which are often aggressive omnivores (Hölldobler and Wilson 1990a; Nie et al. 2023), often benefit from altered or degraded environments (Lach 2021; Siddiqui et al. 2021). They are often highly abundant in their introduced range and can outcompete native ants (Steinbeiss et al. 2009). It has been shown that biochar application often improves ecological conditions for soil microorganisms and fungi (Steinbeiss et al. 2009; Atkinson et al. 2010; Jones et al. 2012), which can promote the food sources of both large and small soil organisms. As a result, we can expect to see subsequent changes in forager communities, such as ants.

In this study, we hypothesised that a short-term increase in ant abundance and apparent morphological changes in ants would result from biochar application to the soil. In addition, we hypothesised that the number of invasive species would be reduced in favour of native species. We investigated soil biochar amendment effects on ant communities and their characteristics using field experiments. Changes in soil physicochemical properties after biochar application were described. We examined ant community presence and abundance and ant species morphological characteristics in response to biochar application. This work fills some knowledge gaps about the ant morphological characteristics as affected by biochar application based on a systematic investigation on ant functional traits and body size.

2 Materials and methods

2.1 Study sites

In the autumn of 2021, a randomised block experiment was conducted in an experimental field located in Kunming, southwestern China. The experimental field was located in the regional red soil at latitude 24°50′36″N, longitude 102°51′59″E, and altitude 1905 m above sea level. The city is characterised by a subtropical highland climate with a Cwb Köppen classification. The monthly 24 h average temperature in Kunming ranges from 8.9 °C in January to 20.3 °C in June, with May to October being the monsoon season and the rest of the year experiencing dry weather. The annual rainfall is 979 mm, and the area receives 2,198 h of bright sunshine annually.

To evaluate biochar effects on ant communities, treatment plots consisting of two replicates, each with a 16 m2 area, were established in September 2021. The two plots (BC plot was with biochar, and CO plot was the blank control) were left unmanaged to allow for spontaneous vegetation growth until the end of the experiment (Additional files 1: Figure S1).

2.2 Biochar

Rice is widely planted. Rice straw is thus readily available, and has been well-studied and frequently-recommended for biochar production. The biochar used in the experiment was produced from rice straw collected from Wujiaying County, Kunming, China (24.8°N; 102.8°E). The feedstock was washed, dried, chopped, and milled to pass through a 100-mesh sieve, and then placed into muffle furnace (SX-4–10, Beijing Ever Bright Medical Treatment Instrument, China). The entire muffle furnace was purged with N2 flow (a purity of 99.99% and a flow rate of 1.5 L min–1), and 30 min later, the furnace temperature was raised to 500 °C at 15 °C min–1 and then kept for 2 h. The obtained biochar show C, H, O, and N contents of 50.69%, 2.79%, 16.91%, and 1.16%, respectively. Its ash content was 38.7%, with specific surface area of 38.3 m2 g−1, total pore volume of 0.079 m2 g−1, and pH of 9.1. The obtained biochar was ground, passed through a 300-mesh sieve (Lieke et al. 2018), and applied to the soil in a 5% ratio to a 20 cm depth with a rototiller. The biochar application ratio of 5% is widely recommended for soil remediation according to various previous studies. Before biochar application, all herbs were manually removed from the two experimental plots. Then, the rice straw biochar was applied to the BC plot soil with a rototiller, and the CO plot soil was also dug in the same way. A PVC barrier was buried vertically 10 cm deep in the soil around the BC plots, with 30 cm exposed aboveground and 50 cm from the traps. An ant escape prevention fluid (talcum powder and 75% ethanol at a 1:1 mass ratio mixture) was evenly applied on the both sides of the barrier (Ning et al. 2019), and the anti-escape solution was applied every three days to avoid cross impact among different plots.

2.3 Soil sampling and vegetation survey

Soil samples were collected at a 0–20 cm depth each month between 31 September 2021 and 20 October 2022. The soil samples were gently ground to 2 mm and their gravimetric moisture contents were determined after oven-drying at 105 °C for 24 h. Soil moisture content was calculated and expressed in g kg−1. Soil pH was measured using a benchtop pH meter in a soil/water solution (1:2.5), after being filtered through a 0.50 μm PTFE filter. Meteorological parameters (ground humidity and temperature) were recorded for a continuous week every first week of a month.

Plant growth and diversity were also monitored in the experimental plots. Microclimate, in particular, habitat complexity (herb cover, height of herbs, and abundance of trees and shrubs) is one of the factors that determine ant abundance and morphological characteristics. Another indicator worth noting is the litter under vegetation, which can select and drive ant leg length and movement ability. Specifically, we recorded 5 vegetation indicators (herb height, species, shrub diversity, arbor diversity, and litter) on a monthly basis. The vegetation survey was conducted using both visual estimation and point intercept methods, centred around the sampling plot with a 1 m radius (Vasconcelos et al. 2008).

2.4 Ant sampling

Ants were sampled monthly from 31 September 2021 to 20 October 2022. The sampling method was 7-d pitfall trapping ( Mahon et al. 2017). Traps were opened on the first day, and the samples were collected on the seventh day. Pitfall traps were constructed using 50 mL plastic tubes (r = 3 cm) completely buried in the soil and filled with 20 mL of propylene glycol as an insecticide and preservative (Luo et al. 2022). Four traps were used in each plot. The control plot was labelled as the CO plot, whereas the biochar-treated plot was denoted as the BC plot. After sampling, the traps were returned to the laboratory for further analysis. All the captured ants were collected from the traps and preserved in 70% alcohol (Helms et al. 2020). Prior to the experiment initiation, we observed three ant nests on the soil surface around the sample plot (with a 50 m radius), the nests were not cleared during the experiment, and no new nests were found.

Captured ants were identified to the species level using dichotomous keys developed by Hölldobler and Wilson (1990a, b) and Bolton (1994) (Hölldobler and Wilson 1990a; Bolton 1994). Morphological identification was based on the references of “A new general catalogue of the ants of the world” (Bolton 1994), and “Chinese Ants” (Wu and Wang 1995). Ant morphological characteristics were observed using a Nikon SMZ1500 stereoscopic zoom microscope (Nikon, Tokyo, Japan). All the specimens were stored at the Yunnan Provincial Key Laboratory of Soil Carbon Sequestration and Pollution Control.

In this study, we focused on 13 ant traits that, based on previous research, have been shown to correlate with their natural history and ecology, and are responsive to environmental changes. We followed the guidelines proposed by trait selection and measurement reference (Gibb et al. 2015; Moretti et al. 2016). Five individuals per species were measured for all 13 traits. We measured WL, which is often used as a surrogate for TL (Tiede et al. 2017). This trait is indicative of insect metabolism, thermal tolerance, foraging duration, and respiration rate. Head length (HL) is an indicator of diet and food preferences. The head width (HW) was measured to determine the gap size through which workers could pass. Procoxal length (PL), metafemoral length (MeL), and tibial length (TiL) were also measured, as they relate to foraging speed and mobility. According to the grain-size hypothesis, these traits are related to body size and habitat complexity. Eye length (EL) is indicative of feeding strategy, daily foraging time, and habitat perception, whereas eye width (EW) is indicative of foraging behaviour and activity times. Eye position (EP) was also measured because it is related to the hunting method or occupied habitat components (Gibb and Parr 2013). Mandible length (MaL) and width (MaW) are related to diet, whereas scape length (SL) is indicative of ant sensory abilities.

To examine the relationships among traits and account for covariation among different traits, we conducted a preliminary principal component analysis and pairwise correlation analysis of all trait pairs. These analyses revealed that all traits were highly correlated and covaried with size as characterised by WL. Therefore, we used the log-transformed WL as the core trait, and the other variables were divided by WL for further comparison. Given the general ant size lognormal distribution, all measurements were log (x + 1) transformed prior to analysis (Zhang et al. 2022).

2.5 Site description before biochar application

No significant differences (P < 0.05) were observed between the two plots in soil physicochemical properties, such as pH, temperature, water content, total organic carbon, and plant species (Additional files 1: Table S1), prior to biochar application. Furthermore, no significant differences (P < 0.05) were observed in ant colony species richness or diversity between the two plots (Additional files 1: Table S1). We selected three common ant species from the colony and measured the morphological indicators of five workers at each sampling point (13 indicators in total). No significant differences (P < 0.05) were observed in the morphological indices of the same species between the two plots.

2.6 Statistical analysis

We used the Kruskal–Wallis nonparametric test to compare soil parameters in different plots. Before statistical comparison, we performed post-hoc tests using the Behrens–Fisher test. A one-way independent analysis of variance was performed on ant abundance in the different treatments, coupled with Dunnett’s post-hoc tests when necessary. All statistical analyses were performed using IBM SPSS 26.

We conducted analyses for the investigated three ant species. For S. invicta, we used generalized linear regression (GLM) to model how species abundance varied with the environmental conditions in BC and CO plots. Because species numbers for the C. nuda and F. japonica were too few to support detailed analytical models of species richness, we analyzed driving factors using generalized linear regression techniques with a binomial error distribution (logistic regression, GLM). Variance inflation factors (VIF values) were used for all soil and environmental variables to exclude multicollinearity. We tested all possible model combinations according to Bayesian Information Criterion (BIC), following the standardized protocols. Pearson correlation analysis was performed between ant morphological characteristics and environment indicators. Significance was set at P < 0.05 in all analyses.

3 Results

3.1 Biochar addition effects on soil properties

Biochar application effects on soil over a 12-month period are shown in Fig. 1. Temperature, moisture content, pH, and vegetation are representative indicators, because they not only well reflect the significant changes after the use of soil biochar, but also directly lead to sensitive responses of ants (such as ant community composition, species richness, and individual morphology). Biochar application resulted in an increase in soil pH throughout the experiment (Fig. 1a), soil moisture content (Fig. 1b) and soil temperature (Fig. 1c). In addition, herb diversity significantly increased (P < 0.05) with biochar addition (Fig. 1d). Compared with CO plot, the application of biochar promoted the increase in pH (32%), temperature (5.8%), and moisture content (24%) in BC plot.

Fig. 1
figure 1

The soil pH, water content, and temperature increased after the application of biochar. The comparison of soil pH (a), soil moisture content (b), temperature (c) and herb diversity (d) in two sample sites (BC and CO) over time (2021.11–2022.10)

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3.2 Ant diversity affected by biochar application

In our study, we successfully collected 2765 ants, of which 2748 ants were accurately identified to the species level. These ants belong to three species, Solenopsis invicta, Formica japonica, and Cardiocondyla nuda, and are classified into three genera and three subfamilies that are commonly observed in Kunming. Notably, Solenopsis invicta is an invasive species from Brazil that is known to be more aggressive than other ant species. Moreover, previous studies have shown that the diversity and abundance of local ant species are suppressed by S. invicta due to habitat occupation and competition for food and other living resources. Our results indicated that overall ant abundance and species diversity were significantly altered at the colony level (P < 0.05) during the one-year observation period following biochar application. Specifically, F. japonica and C. nuda abundances were significantly higher in the biochar-treated plot (P < 0.05), whereas red fire ant abundance (S. invicta) was significantly higher in the control plot (P < 0.05) (Fig. 2). The application of biochar altered the richness of three ant species. Specifically, the abundance of C. nuda and F. japonica increased by 426% and 93%, respectively, while S. invicta experienced a notable decrease in specie richness by 54% after biochar application.

Fig. 2
figure 2

Abundance of three species ants. The comparison of ant abundances of Formica japonica (a), Cardiocondyla nuda (b), and Solenopsis invicta (c) from two sample sites (BC indicates biochar plot and CO indicates control plot) over time (2021.11–2022.10)

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The most supported GLM model (r2 = 0.35, P < 0.01) to explain the abundance of S. invicta included parameters of soil pH (P < 0.01), moisture content (P < 0.01), and average temperature (P < 0.01) (Fig. 3). pH, soil water content, and soil average temperature had the lowest BIC values (BIC = −35), which constituted the optimal explanatory variables of the optimal model.

Fig. 3
figure 3

The correlations between the abundance of S. invicta and soil pH (P < 0.01) (a), soil moisture content (P < 0.05) (b), and soil average temperature (P < 0.01) (c). The gray areas indicate the 95% confidence intervals

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For C. nuda and F. japonica, the most supported logistic regression of GLM for ant abundance included parameters of soil pH and average temperature, with the lowest BIC values. According to the results of the model, higher pH and soil average temperature were associated with a greater abundance of C. nuda and F. japonica (Table 1).

Table 1 Coefficient estimates and diagnostics from binary logistic regression, presenting significant soil variables that explained the abundance of two ant species among BC and CO plots
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3.3 Ant traits affected by biochar application

To ensure CO plot representativeness throughout the experimental period, ants were sampled from four plots with a 50 m radius around the CO plot before (September 2021) and after (September 2023) the experimental period. Our analysis showed no significant differences in 13 ant morphologies around the sampling plot before and after the experiment (P > 0.05), in the surrounding four plots, or in the CO plot (P > 0.05).

Our analyses of 436 ant individuals revealed that 7 F. japonica ant traits and 13 S. invicta ant traits significantly increased after biochar application (Mann–Whitney U-test, P < 0.05, Fig. 4). No significant differences were observed for any C. nuda ant traits during the one-year sampling period (Fig. 4). Regarding F. japonica, our results indicated that seven ant traits (WL, TL, MeL, TiL, MaL, EW, and SL) had the same distribution pattern at both sites (TL was showed in Fig. 4, WL, MeL, TiL, MaL, EW, and SL were showed in Additional files 1: Fig. S2). The ant individuals in the biochar-treated plots exhibited relatively longer or larger ant traits. After applying biochar, F. japonica in the BC plot showed larger total body length (18%), Weber’s length (6.6%), metafemoral length (9.3%), tibial length (13%), eye width (9.4%), mandible length (16%), and scape length (6.3%) (Fig. 4; Additional files 1: Fig. S3).

Fig. 4
figure 4

The comparison of total body length without and with BC application of two species of ants, F. japonica (a) and S. invicta (b). The experiments were carried out during 2021.09–2022.10. The body sizes of F. japonica and S. invicta were bigger in the BC plot than CO plot (c). The body size of C. nuda was not significantly different between BC and CO plots

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A Pearson correlation analysis was carried out between morphological characteristics of ants (F. japonica and S. invicta) and environment indicators (3 soil properties and 5 vegetation indicators). The collinearity of different parameters was excluded before the analysis. These results highlighted the importance of soil properties, particularly pH, soil average temperature and soil moisture content in shaping the morphological characteristics of F. japonica and S. invicta and indicated potential adaptations of the ants to specific environmental conditions.

In Additional files 1: Table S3, the results of F. japonica showed that the total body length (R = 0.712, P < 0.01), Weber’s length (R = 0.384, P < 0.01), metafemoral length (R = 0.398, P < 0.01), tibial length (R = 0.453, P < 0.01), eye width (R = 0.426, P < 0.01), mandible width (R = 0.492, P < 0.01), scape length (R = 0.381, P < 0.01) were correlated positively to the soil pH significantly. Moreover, the total body length (R = 0.286, P < 0.01), and mandible width (R = 0.505, P < 0.01) of F. japonica were correlated to the soil average temperature, and the ant’s mandible width (R = 0.286, P < 0.05) was correlated to soil moisture content.

For S. invicta, the Pearson correlation analysis (Additional files 1: Table S3) showed that total body length (R = 0.373, P < 0.01), Weber’s length (R = 0.335, P < 0.01), head length(R = 0.326, P < 0.01), head width (R = 0.381, P < 0.01), eye position (R = 0.375, P < 0.01), eye length (R = 0.246, P < 0.01), eye width (R = 0.349, P < 0.01), mandible length (R = 0.275, P < 0.01), mandible width (R = 0.225, P < 0.01), and scape length (R = 0.366, P < 0.01) were correlated positively to the soil pH. Eye width (R = 0.251, P < 0.05) was correlated to the soil moisture content, and total body length (R = 0.220, P < 0.05) was also correlated to soil average temperature.

4 Discussion

4.1 Ant traits and their ethology

Species abundance is the most promising parameter for evaluating species adaptability to the environment. In this study, biochar application had contrasting effects on the three species studied. The increased C. nuda and F. japonica abundances highlighted the beneficial biochar effects on these ants. Previous studies have also confirmed that biochar application for soil restoration has several ecological benefits, including altering ant colony species composition and increasing ant species richness (Castracani et al. 2015). Researchers have also found that ants (T. caespitum) tended to move to plots with biochar (Hölldobler and Wilson 1990b). Other soil and compost animals prefer carbon-rich environments. For example, Menzel et al. (2005) showed that C. elegans, when given the choice, actively migrated to humic-enriched feed bacteria (Menzel et al. 2005). In the current study, we applied an ant-escape prevention fluid to restrict ants in the plots, and it was unlikely that ants would have moved across the plots. Thus, the observed increased abundances of F. japonica and C. nuda were attributed to the preferred living conditions of these two ant species, such as wet environment, 6.5–8.0 soil pH range, and high temperature (approximately 35 °C) (Frouz and Jilková 2008).

In contrast, S. invicta thrives in acidic soils and may have experienced significant stress due to the increased soil pH (from 5.0 to 7.5) after biochar amendment (Castracani et al. 2015). In this study, stress significantly reduced S. invicta abundance in the current experiment. S. invicta prefers dry habitats and is sensitive to temperature changes. Previous studies indicated that S. invicta individual reproduction ceases at > 25 °C, and their respiration peaks at approximately 32 °C (Vogt and Arthur 1999). Therefore, the reduction in soil albedo and the increase in soil temperature and moisture content after biochar amendment may have resulted in decreased S. invicta abundance. The increased abundance of local ants (F. japonica and C. nuda) may have benefited, in part, from reduced interspecific competition.

Applying biochar to soils can increase soil temperature (approximately 2–3 °C) (Feng et al. 2021), water content (approximately 13–51%) (Razzaghi et al. 2020), and pH by 46% (Singh et al. 2022), thereby appropriately enhancing soil ecosystem services and promoting effective water retention, soil fertility, and soil organism diversity, including microbes, animals, and plants (Blanco-Canqui 2020; Singh et al. 2022). Our study found that ant communities in biochar-treated sites (wetter and more vegetation) generally had larger body sizes, larger eyes, and longer femurs, and showed an advantageous foraging strategy. Biochar application creates a favorable habitat for C. nuda and F. japonica ants, characterized by optimal conditions (36 ℃ temperature, neutral pH, and increased humidity), resulting in enhanced growth, reproduction, and larger body sizes. Simultaneously, biochar could enrich microbial populations, enhancing the complexity of the omnivorous ant’s food web, leading to increased eye size and antennae length to meet the energy demands of larger body sizes. In S. invicta, larger individuals are selectively favored due to their longer legs, allowing for faster running and elevation of their bodies from the ground. This advantage persists even under conditions that exceed their tolerance threshold, highlighting the invasive species’ adaptability. According to the grain-size hypothesis (Kaspari and Weiser 2002), body size and leg length are positively correlated and determine how ants perceive their environment. Ants with larger body sizes and longer legs can respond to potential barriers in a dense understory, such as plants and litter on the soil surface (Gibb and Parr 2013), which may not directly reflect a beneficial or detrimental effect. However, it should be noted that our one-year observation only considered individual growth due to changed environmental conditions, not biological evolution or long-term biological development.

Eye size of ants has been observed to scale with body size. Specifically, an increase in body size, particularly under dry conditions, results in a proportional increase in eye size (Ibarra-Isassi et al. 2023; Weiser and Kaspari 2006). Previous studies have linked eye size to various ant ecology aspects. For example, subterranean and predatory species tend to have smaller eyes, whereas omnivorous species tend to have larger eyes. In this study, biochar application may have altered the soil food web structure by increasing microorganisms and flora abundance (Jeffery et al. 2022). Thus, changes in food sources and increased body size may have contributed to the enlarged eyes of the ants in the biochar plots.

4.2 Biochar ecological implications

Biochar application to the soil can have beneficial effects on soil fauna (Jeffery et al. 2022; Quilliam et al. 2013). It has been well demonstrated that biochar can promote soil aggregate formation, increase soil structure porosity, and thereby improve soil fauna living conditions. Previous studies have reported that the recalcitrant nature of biochar can provide a habitat for microorganisms that supports soil fauna growth and activity. Therefore, biochar can increase soil fauna biodiversity and activity, and in turn, improve soil health and productivity. The chemicals and persistent free radicals in biochar have adverse effects on some soil microorganisms, such as mycorrhizal fungi, which play critical roles in nutrient cycling and plant growth (Biederman and Harpole 2012). Biochar can also alter the soil pH, which can adversely affect the growth and survival of certain plant species. It should also be noted that even for a given species, different researchers have reported completely opposing results regarding biochar toxicity owing to the different endpoints used in different studies. For example, Sun et al. (2022) observed apparent mortality and weight loss in earthworms (Eisenia fetida) after biochar application to soils (Sun et al. 2022), whereas Khan et al. (2019) found that biochar application increased E. fetida cocoon production, and growth and reproduction rates (Khan et al. 2019).

Based on the above discussion, we drew attention to the following two issues that should be considered when evaluating the ecological role of biochar.

(1) In addition to individual organism physiological responses to biochar application, the species ecological function should be properly evaluated. The most commonly used endpoints to evaluate biochar application effects in soil include species abundance, individual weight, mortality, and fecundity (Castracani et al. 2015; Chen et al. 2022). However, these parameters may not fully reflect the function of these species in soil systems and increased individual weight may not always be beneficial for species competitiveness. This study investigated biochar application effects on ant functional properties. Leg length, eye size, head size, and mandible size (length and width) are related to ant movement speed, foraging strategy, habitat perception, and obstacle crossing, respectively. Significant morphological changes were observed after one year of biochar application, however, the change in individual physiological traits was not always beneficial to their ecological functions. For example, if ant (such as F. japonica) body size or leg length increases, their preferred foraging habitat may shift toward the surface layer with vegetation litter because ants with larger body sizes can cross obstacles more easily. However, F. japonica underground nesting behaviour may be hindered by increasing the time and distance spent foraging. Therefore, species ecological function affected by biochar may be more relevant when investigating its environmental effects.

(2) Biochar environmental impact should be evaluated based on overall biocenotic responses, not just individual species. Most toxicity studies have focused on single species detailed physiological responses. However, adverse effects on one species do not necessarily indicate an overall adverse effect at the biocenosis or ecosystem level. Similarly, the observed enhancement of one species was always accompanied by the suppression of one or more other species. In this study, biochar application resulted in a significant increase in ant community structure species composition and richness, not only reflected in the total species richness, but also showed a significant increase in F. japonica and C. nuda richness. However, S. invicta richness significantly decreased. Biochar application created more favourable living conditions for the native species, suggesting a generally balanced ecological system. Future studies should include more parameters to reflect species persistence or ecological system stability affected by biochar application. In other words, degraded systems that favour aggressive omnivorous species invasion with native, more specialised species suppression can be partially restored to pre-degradation conditions and natural biocenoses. Subsequent studies are required to determine whether this restoration success translates into increased ecosystem resilience, presumably coupled with increased productivity.

It should be noted that this one-year pilot study only examined transient morphological changes in ants, which is not long enough to be reflected in gene expression. Epigenetic modifications and even epimutations may occur and drive this adaptation (Steinberg 2023), which is worth studying in detail in soil invertebrates. Alterations in the soil environment, such as biochar leaching or ageing, can return the systems to baseline conditions. It is still unknown how long the biochar function can be maintained in the system and whether ant function genetic modification is possible via biochar. Therefore, further studies are warranted.

5 Conclusions

This study is the first to assess biochar amendment effects on aboveground soil mesofauna (community diversity, species richness, and ant functional traits) in a field experiment. Soil water content, pH, and mean soil temperature physicochemical properties, which are important parameters controlling ant diversity and community structure, increased significantly after biochar application. Our observations suggested that ant functional traits were significantly altered after biochar application. We emphasise that these trait changes mostly reflect adaptation to the changed environment but not the competitiveness of one species over another. We also observed that biochar application promoted Cardiocondyla nuda and Formica japonica abundance, but decreased that of Solenopsis invicta. Thus, it is helpful to evaluate biochar impact from an ecological perspective. We also emphasise that our conclusions are based on short-term observations, especially when considering biochar ageing and leaching in the soil system, and the observed trait changes may not reflect genetic change or epigenetic adaptations. Biochar environmental effects must be evaluated on a broader ecological basis over longer periods. Nevertheless, the beneficial short-term biochar application effects on ant communities open up promising prospects for successful degraded soil restoration.