Amoebae MPN
Control showed in total, 3,243 individuals per g−1 soil-dry-weight (i g−1 sdw), and CI showed 1023 i g−1 sdw at 9-cm depth (Table 1). Morphotypes VI (Amoebidae: 225 i g−1 sdw) and VII (Leptomyxidae: 151 i g−1 sdw) were dominant at 9-cm depth from CI. Morphotype II (Platyamoeba: 151 i g−1 sdw) was dominant at 9-cm depth in CII. Morphotypes VII (191 i g−1 sdw) and III (Hartmannella: 112 i g−1 sdw) were dominant for CIII (Fig. 2a; x2, P = 0.05; Table S3).
Table 1
Species richness (SR) and most probable number (MPN) of soil free-living amoebae in Unplanted (control) sandy soil (A), N/AMF planted sandy soil (B), and AMF-planted sandy soil (C). Columns (C-I, C-II and C-III) corresponding to compartments of the mycorrhizospheric-box. The species richness is the account of different species recorded in the three compartments rather than the sum of each compartment. The total number of species richness by layer (SRT) and the total MPN by layer I are also included. Row headings: 3-cm, 6-cm and 9-cm are the sampling depths. Row heading: Total by MPN is the sum of quantities reported in the 3 depths (3, 6 and 9-cm)
|
Control
|
N/AMF
|
AMF
|
|
CI
|
CII
|
CIII
|
CI
|
CII
|
CIII
|
CI
|
CII
|
CIII
|
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
SR
|
MPN
|
3cm
|
12
|
266
|
12
|
222
|
10
|
662
|
31
|
703
|
22
|
476
|
10
|
618
|
31
|
870
|
41
|
549
|
16
|
288
|
6cm
|
15
|
188
|
16
|
330
|
10
|
132
|
44
|
303
|
26
|
139
|
14
|
351
|
32
|
500
|
48
|
691
|
17
|
474
|
9cm
|
19
|
569
|
9
|
349
|
12
|
524
|
25
|
674
|
23
|
472
|
19
|
214
|
41
|
69
|
54
|
427
|
29
|
605
|
SR by compartment
|
26
|
|
20
|
|
18
|
|
60
|
|
33
|
|
23
|
|
47
|
|
57
|
|
29
|
|
SRT
|
27
|
65
|
73
|
Total by column MPN
|
|
1023
|
|
902
|
|
1318
|
|
1679
|
|
1087
|
|
1183
|
|
1439
|
|
1667
|
|
1368
|
N/AMF presented in total, 3,950 i g−1 sdw (Table 1). Root-zone showed a MPN = 1,679 i g−1 sdw. CI showed 703 i g−1 sdw at 3-cm depth (Table 1). Morphotype VIII exhibited 603 i g−1 sdw at 9-cm depth for CI. Morphotype VIII dominated at 3-cm and 9-cm depth from CII. Morphotype IV (Saccamoeba: 329 i g−1 sdw) at 3-cm from CI. Morphotypes II and III showed 191 and 226 i g−1 sdw, respectively, from CIII at 3-cm depth (Fig. 2b; x2, P = 0.05; Table S3).
AMF treatment showed 4,473 i g−1 sdw (Table 1). Although root-zone displayed 1,439 i g−1 sdw, and CII showed 1,667 i g−1 sdw, in total. CI showed 870 i g−1 sdw at 3-cm, in which morphotypes III, VI, X (Tetramitus) and VIII registered 224, 149, 118 and 114 i g−1 sdw at 3-cm depth Morphotype X was dominant at all depths (112 to 217 i g−1 sdw) from CII, and morphotype V (Mayorella) exhibited 191 i g−1 sdw at CIII (Fig. 2c; x2, P = 0.05; Table S3).
Distribution And Species Richness
Control treatment registered 27 species (21 genera from 9 families; Table S4). Species richness along depths stayed constant, except for CI (Table 1). One species was exclusive in this treatment.
N/AMF treatment recorded 65 species at 30 genera from 19 families (Table S4). CI presented 44 species at 6-cm depth while CII 26 species were recorded at same depth. In contrast, CIII showed the fewest species richness (Table 1). Seventeen species were registered exclusively in this treatment.
For AMF treatment, 73 species were registered, which consisted in 34 genera from 18 families (Table S4). CI showed 41 species at 9-cm depth. CII registered 54 species at 9-cm depth (x2 P=0.05; Table 1). This treatment showed 20 species as exclusive.
Proportion And Diversity Trophic Groups
Regardless sampled depth and compartment, the most abundant trophic group was bacterivorous (B) amoebae (>60%) at all treatments (Fig. 3a,b,c). the fungivorous (F) trophic group only was found at 3-cm depth from AMF treatment, when compared to Control and N/AMF treatments in which the proportion of this trophic group was about 20% (Fig. 3b). In general, protozoa-eater amoebae (P) was registered in all treatments and compartments, excepting at CIII from AMF treatment at any sampled depth (Fig. 3c).
For control treatment, trophic diversity showed highest values for H’ and D’ indexes (1.2 and 0.9, respectively) at CI and CIII (Table S5), but the highest value of H’=1.4 was recorded from CIII at 9-cm depth (Fig. 3d, Table S6). In contrast, N/AMF treatment showed highest H’ and D’ indexes (1.3 and 0.75, respectively) at CI and CIII, which were similar to those indexes estimated for AMF treatment (H’=1.4 and D’ =0.6) at CI (Table S5). Regarding sampled depth, N/AMF showed values of H’=1.4 at 6-cm depth, and D’=0.7 at 3-cm depth, both from CI. For AMF treatment the highest value of H’ (1.2) was achieved at 3 and 9-cm depth from CI, whereas for 6 and 9-cm the H’ value was similar (1.2) at CII (Fig. 3d; Table S6).
Beta diversity
Species compositional change (Whittaker descriptor) among compartments from N/AMF treatment was βw = 2; whereas for treatments Control and AMF the βw values among compartments, ranged between 1.2 and 1.4, respectively. In N/AMF treatment, βw was of 1.2 among 3 and 6-cm depth from CIII; but for CI the βw was of 1.3 at between 6 and 9-cm depth (Fig. 4). For AMF treatment, βw value was of 1.3 between 3 and 6-cm depth, for CIII (Fig. 4a). Overall, the βw values for the control treatment were as follows: CIII > CII > CI; for N/AMF treatment values were CIII > CI > CII; and for AMF treatment the Bw values were CIII > CI > CII (Fig. 4a; Table S7). Regardless sampled depth and microcosm compartments, Bw values for the treatment effect were as follows: N/AMF (2.0) > AMF (1.8) = Control (1.8) (data not shown).
Beta diversity with specific gradient (sampled depth)
Cody index (βC) from Control treatment ranged from 7 to 5 among the three sampled depths from the three compartments (Fig. 4b). The βC value of CI from treatment N/AMF was of 16, but at their adjacent compartments (CII and CIII) showed lower values (Fig. 4b). In AMF treatment, βC values ranged from 8 to 11, and the highest value was recorded at CII among 6 and 9-cm depth (Fig. 4b; Table S7).
Control treatment showed a beta turnover index (βT, Wilson & Schmida) of 0.5 between 3 and 6-cm depths, at CIII. N/AMF recorded a βT=0.6 in average among 6 and 9-cm depth at CI; whereas for AMF treatment the highest value of βT (0.4) was achieved from 3 and 6-cm depth, at CIII (Fig. 4c).
Cody index βcO from Control treatment was of 0.5 in average, from 3 and 6-cm depth, at CIII. For treatment N/AMF the βcO value was 0.6 between 3 and 6-cm depth, at CIII; whereas for treatment AMF the βcO value was of 0.5 between 3 and 6-cm depth, at CIII (Fig. 4c; Table S7).
Bacterivorous Amoebae Beta Diversity
Bw value for bacterivorous amoebae from Control treatment, was 1.7, which was recorded at CIII, between 3 and 6-cm depths; whereas for N/AMF and AMF treatments the highest value of βw was 1.4 and 1.3 from 3 and 6-cm and 6-9-cm depths, at CIII (Fig. 4d; Table S8).
Regarding specific depths gradient, bacterivorous beta diversity showed the following highest values: Control, βC = 7 between 6 and 9-cm depth, at CI; N/AMF, βC = 16 among 3 and 9-cm depths, at CI; and AMF, βC = 11, between 6 and 9-cm depth, at CII (Fig. 4e; Table S8).
The βT index value for Control, N/AMF and AMF treatments were araund of 0.5 from the same 3 and 6-cm depths, at CIII (Fig. 4f). Moreover, βCO values for N/AMF treatment was of 0.6, while for AMF and Control treatments, this value was around of 0.5, at CIII (3 and 6-cm depth) (Fig. 4f; Table S8).
AMF mycelium length and Pearson Correlation Coefficient with MPN and species richness of amoebae
Form AMF microcosm, the longest AMF-mycelium length (19 cm) was achieved in CI at 9-cm depth; however, for CII showed the longest mycelium length (9.5 cm) was recorded at 6-cm depth, whereas for CIII, the longest mycelium length (6.5 cm) was registered at 6-cm depth. No AMF-mycelium was achieved from C (Non-planted) neither N/AMF microcosms (data not shown).
By considering data from AMF microcosm, the CI showed a negative correlation between the NMP of amoebae with the AMF-mycelium (ρ = -0.9; 81.7% determination), at 9-cm depth. In contrast, the correlation of mycelium length with amoebae species richness was positive 0.4 (14% determination). Species richness and length mycelium correlated ρ = 0.9 (80% determination) recorded in CIII (Fig. 5).
Similarity Analysis
Overall, Sørensen analysis revealed a core community composed mostly by bacterivorous; however; amoebae community composition changed when AMF was present (Fig. 6, B = red circles) when compared to C and N/AMF microcosms (Fig. 6).
Amoebae species composition was dissimilar at microcosms which plants were established (N/AMF and AMF); furthermore, this dissimilarity was observed among their own compartments (Control microcosms) (Fig. 6). Species composition from the microcosms N/AMF showed a cluster (CIII-6 and CIII-9, with 98% of similarity); moreover, this cluster shared 68% of their species with the amoebae community registered at CII-9. There was observed N/AMF, CI-3, CI-6 and CI-9 were dissimilarity of their species communities; furthermore, the remaining communities from CII, shared less than 81% of species (Fig. 6).
For AMF microcosm, the compartments CII-3, CII-6 and CII-9 configured a cluster with 87% of similarity, CII-6 and CII-9 shared 100% of their species this subgroup shared 63% their species with the communities from CI and CII at AMF y N/AMF respectably. Subgroup conformed of CIII-3 and CIII-6 (77%), but by adding the CIII-9 they shared 57% of species among these compartments (Fig. 6).
More specifically, bacterivorous communities from three depths shared species among all microcosms (AMF, N/AMF, and Control), if we considered those communities that shared at least 50% of their species, it is possible to found three different groups which are represented in the corresponding dendrogram (Fig S5a). Principal group was integrated by the communities from all treatments, but from this large group two 2 subgroups were detected. Other group was chained between communities founded from the AMF microcosms at the same compartment (CIII), regardless the sampling depth (Fig S5a). In contrast, communities registered for the Control microcosm had 62% similarity between CII and CIII, mainly at 3 and 6-cm of depth (Fig S12a).
Protozoa-eater amoebae community was formed four groups: the first one involves the control from three compartments with 100% similarity. The third and large group were integrated by AMF and N/AMF microcosms with 62% similarity (Fig S5b); moreover, AMF microcosms (CI) at 3 and 6-cm depth, showed 98% of similarity (Fig S5b).