3.1. Impact of mining activity on soils
A detailed description of physical and chemical properties, and chemical elements contents of the soil and substrate from the sampling areas is shown in Table 1. The soils from the urban ruins area, the Exp 1 and off-mine area were moderately alkaline (approx. pH= 8.3), while the soil of the Exp 2 area was slightly alkaline with a pH value of 7.8. In contrast, the substrate sampled from the dump in the sampling site J (Exp 2) showed an acidic pH value of 5.5. The EC and TDS values for soils in Exp 2 were higher compared to the other sampling areas (5.46 mS cm-1 and 1.49 ppt, respectively), whilst the unexploited area had the lowest EC and TDS values (0.48 mS cm-1 and 0.21 ppt, respectively).
The lowest concentrations of Ca and Mg ions (2.10 ± 0,10% and 0.78 ± 0.08%, respectively) were recorded at the Exp 2 (sampling site HKI), with low content of Na and K in the sampling site J (0.27 ± 0.03 % and 0.5 ± 0.04 %, respectively). The value of S of substrate from sampling site J was higher (8.40 ± 0.40 %) compared to the rest of the studied areas, which ranged from 0.15 to 2.80 %. The total C and P values were low in most areas, ranging from 5.47 to 7.10 % for total C and 0.12 to 0.18 % for P, except in the urban ruins area where P concentration slightly increased (0.43 ± 0.04 %).
The Exp 2 hadsignificantly higher concentrations of Fe, Mn, Pb, Zn and Ag compared to the off-mine area. Additionally, no Ag ions were detected in soil samples from the non-impacted area. In particular, the sampling site HIK of the Exp 2 (Fig 1d) had the highest Cu concentration (726 ± 68 mg kg-1) compared to the off-mine area (51 ± 10 mg kg-1) and the other sampling sites within the mine, which ranged from 182 to 248 mg kg-1. Comparable concentrations of Cr were found in the urban ruins and Exp 1 (70 ± 14 and 64 ± 13 mg kg-1 respectively). V was only detected in the Exp 1 and off-mine area, but Exp 1 showed the highest concentration levels (173 ± 26 vs. 138 ± 21 mg kg-1). Sb was only found in Exp 1 and Exp 2, with similar concentrations in Exp 1 and sites H,I and K of Exp 2 (180 ± 18 and 187 ± 19 respectively), and increasing to 214 ± 21 at sampling site J in the latter area.
The non-impacted area had the highest Sr concentration (301 ± 30 mg kg-1), while Exp 2 had the lowest (81 ± 16 mg kg-1). Ba contents in soil samples varied between 446 to 622 mg kg-1 in both the mine and off-mine areas, but the highest concentration of this chemical element was found in the substrate from the dump (1300 ± 100 mg kg-1). Cd ions were only detected in Exp 2, and there were no differences in Ni concentration among the sampling sites.
Table 1. Physical and chemical properties and chemical elements contents of soils and substrate samples from Paramillos de Uspallata mine and an area outside of mine.
Area
|
Urban ruins
|
Exp 1
|
Exp 2
|
Off-mine
|
# Sampling site
|
A, B, C
|
D, E, F, G
|
H, I, K
|
J
|
L, M, N, O
|
pH
|
8.4
|
8.3
|
7.8
|
5.5
|
8.3
|
%H
|
2.64 ± 0.14
|
2.57 ± 0.60
|
2.40 ± 0.10
|
2.33 ± 0.20
|
2.53 ± 0.19
|
EC (mS/cm)
|
0.59
|
1.33
|
5.46
|
3.57
|
0.48
|
TDS (ppt)
|
0.29
|
0.67
|
1.49
|
1.78
|
0.21
|
Total C (%)
|
7.18 ± 0.47
|
7.31 ± 0.60
|
7.10 ± 0.25
|
6.69 ± 0.42
|
5.47 ± 0.79
|
Total P (%)
|
0.43 ± 0.04
|
0.18 ± 0.02
|
0.16 ± 0.02
|
0.12 ± 0.01
|
0.12 ± 0.01
|
Ca (%)
|
6.20 ± 0.30
|
3.20 ± 0.20
|
2.10 ± 0.10
|
2.40 ± 0.10
|
5.40 ± 0.30
|
Mg (%)
|
1.37 ± 0.07
|
1.52 ± 0.08
|
0.78 ± 0.08
|
0.35 ± 0.04
|
1.65 ± 0.08
|
Na (%)
|
0.81 ± 0.08
|
1.10 ± 0.06
|
0.97 ± 0.07
|
0.27 ± 0.03
|
0.89 ± 0.09
|
K (%)
|
2.40 ± 0.10
|
2.45 ± 0.10
|
2.60 ± 0.10
|
2.00 ± 0.10
|
2.30 ± 0.10
|
S (%)
|
0.31 ± 0.03
|
0.71 ± 0.07
|
2.80 ± 0.10
|
8.40 ± 0.40
|
0.15 ± 0.02
|
Mn (%)
|
0.46 ± 0.05
|
0.53 ± 0.05
|
1.85 ± 0.12
|
2.00 ± 0.10
|
0.11 ± 0.10
|
Fe (%)
|
5.20 ± 0.30
|
5.70 ± 0.30
|
8.15 ± 0.30
|
7.30 ± 0.40
|
4.20 ± 0.20
|
Pb (%)
|
0.43 ± 0.04
|
0.53 ± 0.05
|
1.17 ± 0.75
|
1.00 ± 0.10
|
Det < 0.005
|
Zn (%)
|
0.16 ± 0.02
|
0.79 ± 0.08
|
2.47 ± 0.08
|
2.80 ± 0.10
|
0.020 ± 0.002
|
Ag (mg Kg-1)
|
101 ± 14
|
117 ± 15
|
339 ± 34
|
388 ± 39
|
ND < 50
|
Cu (mg Kg-1)
|
182 ± 18
|
248 ± 25
|
726 ± 68
|
239 ± 24
|
51 ± 10
|
Sr (mg Kg-1)
|
267 ± 27
|
253 ± 26
|
106 ± 15
|
81 ± 16
|
301 ± 30
|
Ba (mg Kg-1)
|
446 ± 45
|
550 ± 55
|
483 ± 49
|
1300 ± 100
|
622 ± 62
|
Cd (mg Kg-1)
|
ND < 50
|
ND < 50
|
Det < 100
|
Det < 100
|
ND < 50
|
Cr (mg Kg-1)
|
70 ± 14
|
64 ± 13
|
ND < 50
|
ND < 50
|
ND < 50
|
Ni (mg Kg-1)
|
57 ± 11
|
49 ± 10
|
39 ± 8
|
48 ± 8
|
32 ± 6
|
Sb (mg Kg-1)
|
ND < 50
|
180 ± 18
|
187 ± 19
|
214 ± 21
|
ND < 50
|
V (mg Kg-1)
|
ND < 50
|
173 ± 26
|
ND < 50
|
ND < 50
|
138 ± 21
|
For each chemical element, the uncertain values correspond to the combined ones. Values of % H y % Total Carbon (TC) represent average ± standard deviation. EC: Electric Conductivity, TDS: Total Dissolved Solutes. ND: Not detected value, below detection limit, Det: Detected value. Exp 1: exploitation area 1; Exp 2: exploitation area 2; Off-mine: off-mine area.
3.2. Plant and Symbiotic Mycorrhizal traits
A total of twelve plant families and fourteen plant species were identified across the different areas within the Paramillos de Uspallata mine, including the area outside the mine (Table 2). Some plant families were only found in certain sampling sites (Geraniaceae and Violaceae families in the off-mine area, Cactaceae in Exp 1 and Rosaceae in Exp 2. The Exp 2 exhibited only three families (Poaceae, Malvaceae and Rosaceae) and a total of four plant species. In contrast, the other sampling sites varied from five and six families with twelve plant species.
The majority of plant species were perennial, either native or endemic to the study area. Two species belonging to the Asteraceae family, both listed in the red list of threatened species (categories 4 and 5, respectively), were found: Senecio uspallatensis in Exp 1 and Artemisia mendozana var. paramilloensis in the off-mine area. Furthermore, two exotic annual therophytes, Erodium cicutarium and a non- identified species from Brassicaceae family, were detected in both areas. The urban ruins area, Exp 1 and off-mine area displayed a predominance of hemicryptophytes, followed by chamaephytes, and a few nanophanerophytes and therophytes, mostly in vegetative phenological state. In contrast, hemicryptophytes and chamaephytes were only found in the most impacted area (Exp 2). No plants or AM fungal propagules were detected in the dump (sampling site J). Outside the mine, perennial herbs were abundant, with the presence of some perennial subshrubs.
The unique mycorrhizal association detected both within the mine and the external area was AM type (Table 2, Fig. S1), with predominance of Arum colonisation. Likewise, some roots of Pappostipa speciosa, Junellia uniflora, A. mendozana, and Tetraglochin alata presented the Arum-Paris type (Table 2). The highest levels of AM root colonisation was recorded in the urban ruins area, Exp 1, and off-mine area in most plant species (Fig 2), and in T. alata plants within Exp 2. The majority of P. speciosa plants were colonised by AM fungi, with the exception of a few individuals in Exp 2. There was no detection of AM root colonisation in plants belonging to the Boraginaceae, Malvaceae, Brassicaceae and Geraniaceae families.
The percentages of plants colonized and non-colonized by AM fungi or DSE were calculated for each area in Paramillos de Uspallata mine and the area without mining activity (Fig. 3). The off-mine area and Exp 1 showed the highest proportion of AM plants (80 and 82% respectively), this percentage drops to 62% in the urban area, and declines to 31% in Exp 2. The root colonisation by DSE also was observed in different proportions in all areas (Fig 3, Table S2), except for Adesmia horrida in the Exp 1 that was only colonised by AM fungi. A low level of root colonisation by DSE was observed in the majority of plants within the off-mine area, while a high level of colonisation was found in the Exp 2 (Table S2).
Table 2. Family and plant species (including the origin of each plant species in brackets), number of individual plants, and plant and symbiotic mycorrhizal traits in each study area within Paramillos de Uspallata mine and an area outside of the mine.
Family/species of plants
|
Sampling area (# individuals)
|
Plant traits
|
Symbiotic traits
|
Functional type
|
Life cycle
|
Phenology state
|
Type of Mycorrhiza
|
Type of AM colonisation
|
Poaceae
Pappostipa speciosa (n)
|
Urban (3)
Exp 1 (5)
Exp 2 (8)
Off-mine (3)
|
H
|
PH
|
v
|
AM
|
A-P, A
|
Pappostipa sp. (n)
|
Exp 2 (3)
|
H
|
PH
|
fl, v
|
AM
|
A
|
Boraginaceae
Phacelia sinuata (en)
|
Urban (1)
Exp 1 (1)
|
C
|
PH
|
fl
|
-
|
-
|
Solanaceae
Fabiana patagonica (n)
|
Urban (3)
|
N
|
PS
|
v
|
AM
|
A
|
Verbenaceae
Junellia uniflora (en)
|
Urban (3)
Off-mine (3)
|
C
|
PSS
|
fl
|
AM
|
A-P, A
|
Malvaceae
Sphaeralcea philippiana (en)
|
Urban (3)
Exp 2 (2)
|
C
|
PH
|
fl
v
|
-
|
-
|
Asteraceae
Senecio uspallatensis (en*)
Artemisia mendozana
var. Paramilloensis (en*)
|
Exp 1 (2)
Off-mine (3)
|
N
N
|
PS
PSS
|
fl
v
|
AM
AM
|
A
A-P, A
|
Brassicaceae
Unidentified species (ex)
|
Exp 1 (1)
|
T
|
AH
|
v
|
-
|
-
|
Fabaceae
Adesmia horrida (n)
|
Exp 1 (1)
|
N
|
PS
|
fl
|
AM
|
A
|
Cactaceae
Maihueniopsis glomerata (n)
|
Exp 1 (1)
|
SC
|
PSS
|
fl
|
AM
|
A
|
Rosaceae
Tetraglochin alata (n)
|
Exp 2 (3)
|
C
|
PSS
|
v
|
AM
|
A-P
|
Geraniaceae
Erodium cicutarium (ex)
|
Off-mine (3)
|
T
|
AH
|
fl
|
-
|
-
|
Violaceae
Viola atropurpurea (en)
|
Off-mine (3)
|
H
|
PH
|
v
|
AM
|
A
|
Urban: urban ruins area; Exp 1: exploitation area 1; Exp 2: exploitation area 2; Off-mine: off-mine area. Functional type: H=Hemicryptophyte, C=Chamaephyte, SC=Succulent Camephyte, T=Therophyte, N=Nanophanerophyte. Life cycle: PH=Perennial Herb, PS=Perennial Shrub, PSS=Perennial Subshrub, AH=Annual Herb. Origin: n=Native, en= Endemic, e= Exotic. Phenological state: v=vegetative, fl=lowering. Mycorrhizal types: AM=arbuscular mycorrhiza. No detection of Mycorrhiza: -. AM colonisation type: A=Arum, P=Paris, A-P=Arum-Paris. * indicate endangered plant species.
3.3. Mycorrhizal Fungal Traits
The AM spore density did not show statistically significant differences between the areas impacted by mining activity and the undisturbed area (F = 1.74; p = 0.22) (Fig. 4). However, a notable disparity was found among sampling sites, particularly in Exp 2. The spore density outside the mine and the Exp 1 was 142 ± 56 and 164 ± 32 (mean ± standard deviation) of AM spores per 10 g of dry soil, respectively. In contrast, the lowest values of spore density were found in the urban ruins area and Exp 2 (70 ± 12 and 99 ± 91, respectively). No AM fungal spores were found in the dump (site J) of the Exp 2.
Although no statistically significant differences were found in diversity parameters (S: F= 2.15 and p= 0.16; H index: F= 1.1 and p= 0.39; D index: F= 1.10 and p= 0.39), a higher richness and diversity values were recorded for the unexploited area, the urban ruins area and the Exp 1, in comparison with the Exp 2 (Table 3). The highest value of D index was found in the Exp 2 (0.44 ± 0.23), contrasting with the rest of the areas. Particularly, within this sampling area, the sampling site I (mineral treatment pools, Fig. 1d) had a large number of AM spores, dominated by Entrophospora infrequens, an ancestral and s-strategist AM fungal species (191 out of 203 spores per 10 g of dry soil).
In regards to the AM community composition and relative abundance species, Claroideoglomus etunicatum, a rhizophilic and r-strategist species, was the most abundant across all areas surveyed, with the exception of Exp 2 (Fig. 5). Its relative abundance (%) varied with 26.8% in the off-mine area, 27.4% in the urban ruins, and 24.8% in Exp 1. In Exp 2, the abundance of this AM species decreased substantially to 11.9%, while the AM species E. infrequent reached 35% of the total spores observed, and Acaulospora scrobiculata accounted for 18.3% (Fig. 5). The second most abundant AM species in the urban ruins area and Exp 1 was Funneliformis geosporus with relative abundance values of 20.3 ± 6.7% and 13 ± 2.7%, respectively. In contrast, Diversispora spurca, Funneliformis coronatus and F. geosporus were found in high proportions in the unexploited area (17.9%, 10.5% and 10.1%, respectively). Also, the edaphophilic and k-strategist species D. spurca was abundant in both the urban area and Exp 1, but was lower in Exp 2 (1.1%). A few species of the genus Rhizophagus were found inside and outside the mine, with consistent proportions across different areas: 6.9% in the urban area, 9.4% for the off-mine and Exp 2, and 9.8% for Exp 1. The AM species G. macrocarpum was exclusively found within the mine in all areas, with a higher abundance value in the urban area (8.1%) than in both exploitation areas (3.1%). Acaulospora rehmii and Entrophospora baltica, both classified as s-strategist species, were recovered exclusively from Exp 2, albeit in low proportions. In Exp 1, ornamented AM spores were recognised but they could not be assigned to any described species, possibly indicating the presence of a new AM fungal species.
The proportionality of the AM fungal families in each area is shown in Fig. 6a. The Glomeraceae and Claroideoglomeraceae families were dominant for all areas, except for Exp 2, where the Entrophosporaceae and Acaulosporaceae families prevailed. The urban area and Exp 1 showed very similar percentages for the two families (55.2-51.5% of Glomeraceae and 31.9-31% of Claroideoglomeraceae), followed by Diversisporaceae (8.6% for urban area and 11.6% for Exp 1). A similar proportion was found for the Glomeraceae and Claroideoglomeraceae families in the off-mine area (39.8% and 33.5%, respectively), followed Entrophosporaceae family (25.2%). In contrast, Exp 2 was dominated by the Entrophosporaceae family (66.8%), followed by the Glomeraceae (14.2%) and Claroideoglomeraceae (11.7%) families. In addition, the presence of Acaulosporaceae species was higher compared to the non-exploited area (6.7% vs. 0.9%), and it was absent in the remaining areas within the mine. A high diversity of AM families was observed outside the mine (Fig. 5 and Fig. 6a) represented by six families: Glomeraceae, Claroideoglomeraceae, Entrophosporaceae, Acaulosporaceae, Diversisporaceae and Paraglomeraceae. Paraglomeraceae species were detected only in Exp 1 and outside mine.
The proportion of functional groups (%) (Table S1) inside and outside the Paramillos de Uspallata mine is shown in Fig. 6b. The Urban ruins, Exp 1 and off-mine areas showed similarities in functional groups, with the rhizophilic and edaphophilic species being abundant, while ancestral and rhizophilic species dominated the Exp 2 area. Considering all areas within the mine, the AM fungal species classified as ancestral or stress-tolerant were the more abundant, comprising 97,6% of this group across all areas (Fig. S2). Specifically, they were dominant at site I (mineral treatment pools) in the Exp 2 area. In contrast, the lowest proportion of these stress-tolerant AM species was observed outside mine. The proportion of rhizophilic species or ruderals within the different areas of the mine was higher (61.7%) than in the off-mine area (38.3%) (Fig. S2). On the contrary, an inverse result was observed for these functional groups, with the majority of edaphophilic species occurring outside the mine (65.3%), while the rhizophilic species accounting for 34.7%, respectively (Fig. S2).
Table 3. Richness values (S) and Shannon (H) and Simpson (D) indices based on morphological taxonomy of AM spores for each area in Paramillos de Uspallata mine and an area without mining exploitation.
Area
|
S value
|
H index
|
D index
|
Urban ruins
|
10.67 ± 1.86 a
|
1.90 ± 0.22 a
|
0.18 ± 0.03 a
|
Exp 1
|
12.67 ± 2.02 a
|
1.98 ± 0.13 a
|
0.17 ± 0.02 a
|
Exp 2
|
7.33 ± 1.45 a
|
1.28 ± 0.54 a
|
0.44 ± 0.23 a
|
Off-mine
|
11.25 ± 0.75 a
|
1.84 ± 0.18 a
|
0.24 ± 0.07 a
|
Mean ± standard error values are reported. No significant differences were found after ANOVA analysis. The same letter indicates no significant difference (p ≤ 0.05) between areas according to Tukey’s test. Urban ruins: urban ruins area; Exp 1: exploitation area 1; Exp 2: exploitation area 2; Off-mine: off-mine area.
3.4. Relationships between physicochemical soil properties and mycorrhizal traits
The PCA was performed to understand the spatial distribution of physicochemical parameters of soils and mycorrhizal traits, as well as to assess the respective contributions of these results to the observed variations among the studied areas (Fig. 7). Within its first two components, it encompasses 86.9% of the variability, with the Principal Component 1 (PC1) and the Principal Component 2 (PC2) explaining 66.3% and 20.6%, respectively. The results revealed a clear separation among the areas, especially for Exp 2, positioned along the PC1 axis. The variables contributing most to this separation included certain physicochemical parameters such as EC and TDS, and various HM (Sb, Zn, Fe, Cu, Mn, Pb and Ag) and other chemical elements (S, K). The urban ruins area exhibited a significant association with the total P content. The off-mine area was primarily characterised by mycorrhizal richness and the concentration of Sr and Mg to a lesser extent. The Exp 1 was associated with the abundance of AM spores, and the chemical elements V, Ba and Na. Finally, the Exp 2 was separated from the remaining areas, and it was explained by the HM content in soils (Pb, Ag, Mn, Zn, Fe, Sb, Cu) together with other chemical elements (K and S), and with the soil physicochemical variables TDS and EC. In addition, AM fungal richness was inversely related with Exp 2.
The dbRDA analysis identified the soil parameter EC as a significant environmental variable (p= 0.04) explained by the axis 1 with 81.3%, and closely linked to the abundance of the AM species E. infrequens in the Exp 2 (Fig. S3)