Temporal effects of urine and carbon inputs on bacterial biodiversity
The mean ASV richness of overall prokaryote communities declined over time for all three treatments, by maxima of 18%, 16%, and 23%, in the N-only, N + C1, and N + C2 treatments, respectively, after 14 days (T3) in each case (Fig. 1a). Richness recovered to 6.7% higher, 4.5% higher, and 1.7% lower than initial values after 112 days (T6) in the three respective treatments. The mean evenness of prokaryote communities changed little in the N-only treatment, declining by at most 1.3% after 14 days (T3), compared to 8.4% after 14 days (T3) in N + C1 and 9.9% after 28 days (T4) in N + C2 (Fig. 1b). Mean evenness recovered to within 1.8% of initial values after 112 days (T6) in all three treatments. The mean abundance of prokaryote communities in the N-only treatment was lower at all time points than initial values, by a maximum of 60% after 14 days (T3), and 18% lower than initial values after 112 days (T6). Mean abundance of prokaryote communities in the N + C treatments were lower than initial values at several time points (1, 7, and 28 days in N + C1; 1 and 14 days in N + C2), by smaller margins than in the N-only treatment, but higher than initial values at all other time points, by up to 59% after 56 days (T5) in N + C1 and 26% after 112 days (T6) in N + C2.
Significant negative cubic temporal trends were detected for relative ASV richness and relative evenness of overall prokaryote communities (F87 = -5.23 to -4.69, p ≤ 0.001; Online Resource 1), indicating asymmetric temporal patterns of richness and evenness decline and recovery, but no significant temporal trends were detected for proportional abundance. The relative evenness of overall prokaryote communities in the N + C2 treatment was significantly lower than that of the N-only treatment (F12 = -3.26, p = 0.007), but no significant differences between N-only and N + C treatments were detected for relative ASV richness or relative proportional abundance.
Biodiversity metrics for individual prokaryote taxa most commonly reached maximal divergence from T0 values after 14 days (T3; 39 combinations of bacterial taxon, treatment, and metric; Online Resource 2, Fig. S1). ASV richness trends per bacterial taxon were similar to that of the overall bacterial dataset (Fig.s 2; Online Resource 2, Fig. S1 a). In the N-only treatment, the mean richness of most taxa declined after one day (T1), increased again after 7 days (T2), then declined again to maxima of 31% (Betaproteobacteria) to 53% (Deltaproteobacteria) below initial values after 14 days (T3), before increasing again from 14 to 112 days (T3-T6). Richness at the experiment end was higher than initial richness for four taxa, by 11% (Betaproteobacteria) to 29% (Bacteroidetes), and lower than initial richness for seven taxa, by just 1% (Actinobacteria, Deltaproteobacteria) to 18% (Firmicutes). Carbon inputs reduced the magnitude of richness declines for all taxa. The largest difference between N-only and N + C treatments was observed for Firmicutes, with a maximum decline of 8.7% after 7 days (T1) in N + C2, compared to 47% after 14 days (T2) in the N-only treatment. Significant negative cubic trends were detected for richness of all taxa except Bacteroidetes (F87 = -2.40 to -4.12, p ≤ 0.001; Online Resource 1). Only marginally significant differences between richness levels in N + C treatments compared to N-only were detected, for Firmicutes in N + C2 (F12 = 2.13, p = 0.054), Chloroflexi in N + C1 (F12 = 2.01, p = 0.067), and Betaproteobacteria in N + C1 (F12 = 1.98, p = 0.071).
Relative evenness and abundance trends varied among taxa to a greater extent than the richness trends did, and these changes were larger in the N + C treatments than the N only treatment (Fig.s 2; Online Resource 2, Fig.s S1 b & c). In particular, the relative evenness of Actinobacteria, Bacteroidetes, Firmicutes, Betaproteobacteria, and Gammaproteobacteria all declined over time (and relative to their N-only trends), by 12% for Bacteroidetes and Firmicutes after 14 days (T3) in N + C1 to 31% for Actinobacteria after 14 days (T3) in N + C2, recovering to slightly higher than the initial values after 112 days (T6) only for Bacteroidetes, Firmicutes, and Betaproteobacteria in N + C1. Somewhat larger evenness declines were observed in N + C2 than in N + C1 for each of these taxa except Betaproteobacteria. These same taxa each had substantial increases in abundance over time, with maximal abundance increases of 26% for Firmicutes after 14 days (T3) in N + C1 to 304% for Gammaproteobacteria after 7 days in N + C2. The mean abundances of these taxa reduced again by T6 (112 days) but were still consistently higher than their initial values by up to 103% (for Gammaproteobacteria in N + C1), except for Firmicutes (lower by 13% and 24% respectively in N + C1 and N + C2). Conversely, the mean abundances of Acidobacteria, candidate division WPS-1, Planctomycetes, Deltaproteobacteria, and Verrucomicrobia in N + C treatments each declined over time (and relative to their N-only trends), by 24% for Acidobacteria after 14 days (T3) in N + C1, to 56% for Deltaproteobacteria after 28 days (T4) in N + C2. Evenness trends for these taxa changed little over time or between treatments.
Significant negative cubic evenness trends were detected for Actinobacteria, Bacteroidetes, Firmicutes, Betaproteobacteria, and Gammaproteobacteria (F12 = -2.66 to -4.06, p = 0.009 to < 0.001; Online Resource 1), while significant positive cubic evenness trends were detected for Planctomycetes and Deltaproteobacteria (F12 = 2.21 and 2.09, p = 0.029 and 0.040, respectively). The relative evenness of Actinobacteria and Betaproteobacteria in both N + C1 and N + C2, and Gammaproteobacteria in N + C2, were each significantly lower than those in the N-only treatment (F12 = -2.78 to -4.28, p = 0.017 to 0.001). In addition, marginally significant differences in relative evenness compared to N-only were observed for Firmicutes in N + C2, Gammaproteobacteria in N + C1, and Bacteroidetes in N + C2 (F12 = -2.01 to -2.06, p = 0.067 to 0.059). Significant positive cubic abundance trends were detected for Bacteroidetes, Betaproteobacteria, Firmicutes, and Gammaproteobacteria (F87 − 99 = 2.21 to 3.33, p = 0.029 to 0.001), whereas significant negative cubic abundance trends were observed for Acidobacteria, candidate division WPS-1, Planctomycetes, Verrucomicrobia, Alphaproteobacteria, and Deltaproteobacteria (F87 − 99 = -2.004 to -4.22, p = 0.048 to < 0.001). Proportional abundances of Betaproteobacteria in N + C1, and Gammaproteobacteria in both N + C1 and N + C2, were significantly higher than those in the N-only treatment (F12 − 99 = 2.82 to 4.23, p = 0.008 to < 0.001). Conversely, proportional abundances of Acidobacteria in N + C2, and both Planctomycetes and Verrucomicrobia in both N + C1 and N + C2, were significantly lower than those in the N-only treatments (F12 − 99 = -2.67 to -3.98, p = 0.009 to 0.002).
Temporal effects of urine and carbon inputs on fungal biodiversity
The mean ASV richness of overall fungal communities declined in all three treatments, by maxima of 26% after 14 days (T3), 41% after 7 days (T2), and 70% after 28 days (T4), in N-only, N + C1, and N + C2 treatments, respectively, partially recovering to 12%, 24% and 40% lower than initial values after 112 days (T6) in the three respective treatments (Fig. 1d). Changes in the mean relative evenness of fungal communities ranged from 6.3% higher than initial values after 7 days (T3) to 18% lower than initial values after 14 days (T4) in the N-only treatment (Fig. 1e). In N + C1 and N + C2, mean fungal evenness was lower than initial values at all time points except for after one day in N + C1, reaching minima of 18% and 38% lower than initial values in each respective treatment after 56 days (T5). The mean abundance of fungal communities was lower than initial values at all time points except 28 days (T4) in the N-only treatment, by up to 37% (after 112 days), and at all time points in N + C1, by up to 88% after 28 days (T4). In contrast, mean proportional abundance in N + C2 was higher than initial values at all time points except 7 days (lower by 54%), by up to 108% after 14 days (T3), and by 34% after 112 days (T6). A significant negative cubic trend was detected for fungal ASV richness (F87 = -4.23, p < 0.001; Online Resource 1). A significant quadratic trend was detected for fungal relative evenness (F87 = 3.32, p = 0.001). Fungal richness in N + C2 was significantly lower than that in the N-only treatment (F12 = -2.45, p = 0.030). No significant differences between treatments were detected for relative evenness or abundance trends.
Biodiversity metrics for individual fungal taxa most commonly reached maximal divergence from T0 values after 28 days (T4; 35 combinations of fungal taxon, treatment, and metric; Online Resource 2, Fig. S2). Fungal ASV richness trends were more variable between taxa and treatments than those for bacterial taxa (Fig. 3; Online Resource 2, Fig. S3a). In the N-only treatment, the mean richness of Eurotiomycetes, Agaricomycetes, and Glomeromycota at all time points after T0 were always lower than initial values, by up to 60% for Glomeromycota after 14 days (T3). All other taxa had richness values that were variably lower and higher than initial values at different time points, with the largest increase at any time point (102%) observed for Mortierellomycota after 28 days (T4). The largest richness declines in N + C treatments were observed for Chytridiomycota and Glomeromycota, by 88 to 92% after 28 days (T4), as well as Dothidomycetes and Sordariomycetes by 78% after 28 days (T4) in N + C1. In contrast, only one taxon (Tremellomycetes) had clearly higher mean richness in the N + C treatments compared to N-only, with a maximal increase of 304% after 56 days (T5) in N + C2. Richness trends for Eurotiomycetes, Microbotryomycetes, Mortierellomycota, and Mucoromycota were similar in all three treatments. After 112 days (T6), the only taxa with higher mean richness that initial values in N + C treatments were Leotiomycetes in N + C2 (by 19%), Tremellomycetes in both N + C1 and N + C2 (by 190% and 248% respectively), and Mortierellomycota in N + C2 (by 20%).
Significant negative cubic richness trends were detected for Agaricomycetes, Chytridiomycota, Dothideomycetes, Eurotiomycetes, Glomeromycota, and Rozellomycota (F87 = -2.01 to -4.81, p = 0.047 to < 0.001; Online Resource 1). Mean richness was significantly lower in N + C treatments compared to N-only for Chytridiomycota in both N + C1 and N + C2, and Sordariomycetes in N + C1 (F12 = -2.83 to -3.47, p = 0.015 to 0.005), while a marginally significant difference was observed for Dothideomycetes in N + C1 only (F12 = -2.17, p = 0.051). Conversely, mean richness of Tremellomycetes was significantly higher in both N + C treatments compared to N-only (F12 = 2.585 and 3.788, p = 0.024 and 0.003).
Relative evenness and abundance trends for fungal taxa were more variable across time points and between treatments than the richness trends (Fig. 3; Online Resource 2, Fig.s S2 b & c). In the N-only treatment, the evenness of most taxa changed little over time. The mean abundances of Dothideomycetes and Sordariomycetes, however, tended to increase over time, respectively reaching 829% and 108% of initial values after 112 days. Conversely, the mean abundances of Eurotiomycetes, Agaricomycetes, and Glomeromycota were consistently below initial values, by up to 72% in each case at various time points.
In the N + C treatments, the clearest evenness declines were observed for Leotiomycetes and Rozellomycota, by maxima of 70% after 14 days (T3), and 80% after both 14 and 56 days (T3 and T5), respectively, in N + C2. Conversely, the mean evenness of Mortierellomycota in the N + C1 and N + C2 treatments increased over time, by maxima of 145% after 28 days (T4), and 101% after 112 days (T6) respectively. The evenness decline observed for Leotiomycetes in the N + C2 treatment was mirrored by a large abundance increase (up to 3,070% after 56 days, T5). Substantial abundance increases in the N + C treatments over time were also observed for Tremellomycetes (up to 1,447% in the N + C2 treatment after 112 days, T6), Mucoromycota (up to 609% in the N + C1 treatment after 28 days, T4), Microbotryomycetes (up to 1,822% in N + C2 after 14 days, T2; but declining to 65% below initial abundance after 56 days, T5), and Mortierellomycota (up to 504% in N + C1 after 7 days, T2). In contrast, the mean abundance of Chytridiomycota in the N + C treatments was clearly lower than values in the N-only treatment, declining by over 90% relative to initial values. Similarly, mean abundances of Eurotiomycetes and Glomeromycota were consistently lower than initial values, by over 90% at some time points, but these trends were similar to those in the N-only treatment. Sordariomycetes and Agaricomycetes mean abundances each increased modestly from 1 to 14 days in the N + C treatments, before declining at subsequent time points (by up to 90% in N + C2 after 56 days; T5).
Significant negative cubic trends were detected for relative evenness of Glomeromycota and Mucoromycota (F87 = -2.72 and − 2.20, p = 0.008 and 0.030, respectively; Online Resource 1). Significant quadratic (but not cubic) trends were detected for relative evenness of Leotiomycetes and Rozellomycota (F87 = 2.37 and 2.82, p = 0.020 and 0.006, respectively) (Table S4). Significantly lower evenness in N + C treatments compared to N-only were detected for Glomeromycota in N + C1, and Mucoromycota in both N + C1 and N + C2 (F12 − 99 = -2.66 to -2.71, p = 0.021 to 0.009), whereas the evenness of Chytridiomycota was higher in N + C1 than in N-only with marginal significance (F12 = 2.16, p = 0.052). Significant cubic trends were detected for Agaricomycetes and Leotiomycetes, as were significant quadratic trends for Chytridiomycota, Eurotiomycetes, Glomeromycota, and Tremellomycetes (F87 = -4.197 to 3.395, p = 0.018 to < 0.001). The mean proportional abundance of Dothideomycetes was significantly lower in both N + C1 and N + C2 treatments compared to the N-only treatment (F12 = -2.554 and − 2.367, p = 0.025 and 0.036, respectively). Conversely, the mean proportional abundances of Leotiomycetes and Microbotryomycetes in N + C2, and Mortierellomycota in N + C1, were each significantly higher than those in the N-only treatment (F12 = 2.351 to 3.635, p = 0.037 to 0.003).
NMDS ordinations showed limited evidence of changing prokaryote community composition over time in the N only treatment, but increasing divergence of prokaryote communities from their initial state with increasing C input levels until at least 28 days (T4) in the N + C1 and N + C2 treatments (Fig. 4a). Samples from 56 and 112 days appeared to show a trend of prokaryote communities converging back towards their initial states. Similarly, fungal communities become increasing divergent from their initial states with time and with increasing C input levels, but to a greater extent than the prokaryote communities (Fig. 4b). Fungal samples from time points after 1 day (T2-T6) were all clearly distinct from those at the start of the experiment (T0-T1) in the N + C1 and N + C2 treatments. The largest C-driven shifts in prokaryote and fungal community Bray-Curtis dissimilarity between consecutive time points evidently occurred between T1 and T2 (1 to 7 days). PERMANOVA tests detected evidence for significantly differing prokaryote community compositions between treatments (F2 = 2.953, p ≤ 0.001) and across times (F1 = 4.058, p ≤ 0.001), and for significantly differing fungal community compositions between treatments (F2 = 3.201, p ≤ 0.001) and across times (F1 = 3.125, p ≤ 0.001; Online Resource 1). PERMANOVA tests between communities detected at the start (T0) and end (T6) of the experiment indicated that neither prokaryote communities (F1 = 3.456, p ≤ 0.001) nor fungal communities (F1 = 2.949, p ≤ 0.001) had returned to their initial composition after 112 days. These tests also detected a significant treatment difference for fungi (F2 = 1.402, p = 0.015) but not prokaryotes (F2 = 1.275, p = 0.127; Online Resource 1).
Over half of the prokaryote ASVs detected in the first samples (T0) were also detected at subsequent time points in each experimental treatment (54–77%), except for 47% after 14 days (T3) in the N only treatment (Fig. 5a). Somewhat larger proportions of prokaryote ASVs were newly detected at each time point throughout the experiment (T1-T6) in the N + C1 and N + C2 treatments (13–43%) than in the N only treatment (6–28%). Conversely, somewhat smaller ranges of the T0 prokaryote ASVs were absent at subsequent time points in the N + C1 and N + C2 treatments (25–41%) than in the N only treatment (31–53%).
The proportions of fungal ASVs changed to a much greater extent than proportions of bacterial ASVs throughout each experimental treatment (Fig. 5b). In the N only treatment, less than half of the T0 fungal ASVs (29–39%) were also detected at subsequent time points, with higher proportions of T0 ASVs not detected (61–76%) than ASVs newly detected (41–57%) at each time point after T0. Carbon inputs resulted in increased losses of fungal ASVs. The proportions of T0 fungal ASVs detected at subsequent time points declined from 39% after one day (T1) to as little as 5.3% after 28 days (T4) in the N + C1 treatment, and from 28% after one day (T1) to 3.1% after 56 days (T5) in the N + C2 treatment. Furthermore, the proportions of fungal ASVs newly detected after seven to 112 days (T2-T6) in the N + C1 and N + C2 treatments (14–35%) were greatly exceeded by T0 ASVs that were absent at these time points (82–97%).
Effects of urine and carbon inputs on prokaryote community function
A total of 230 biosynthesis pathways, 152 degradation/utilisation/assimilation pathways, and 48 precursor metabolite/energy generation pathways were detected using PICRUSt2 (Table 2; Online Resource 2, Fig.s S3-S5). Maximal pathway abundance changes within each treatment, both positive and negative, were typically observed after 14 or 28 days (T3 or T4). Pathway abundances subsequently approached but did not always return to their initial values by 112 days. A similar range of pathways was affected in each treatment, but the magnitude of pathway abundance changes was largest in the N + C2 treatment and smallest in the N only treatment.
Table 2
Changes in prokaryote metabolic pathway abundances after 14 and 112 days (T3 and T6) relative to T0. Values are mean percent abundance changes of pathways per category relative to T0, with the numbers of pathways with increased or decreased abundances shown in parentheses.
Category | Time point | Change | N only | N + C1 | N + C2 |
Biosynthesis | 14 days (T3) | Increase | 19.49 (93) | 131 (77) | 259.97 (88) |
| | Decrease | -8.55 (131) | -13.23 (147) | -17.11 (136) |
| 112 days (T6) | Increase | 39.37 (88) | 40.46 (72) | 35.44 (77) |
| | Decrease | -11.31 (136) | -16.45 (152) | -14.45 (147) |
Degradation/Utilization/Assimilation | 14 days (T3) | Increase | 28.23 (106) | 180.5 (103) | 526.59 (108) |
| | Decrease | -17.56 (40) | -20.52 (45) | -19.2 (39) |
| 112 days (T6) | Increase | 31.46 (105) | 53.48 (101) | 164.27 (108) |
| | Decrease | -20.52 (41) | -16.53 (47) | -15.28 (39) |
Precursor Metabolite and Energy Generation | 14 days (T3) | Increase | 14.16 (28) | 48.15 (28) | 78.61 (29) |
| | Decrease | -18.3 (20) | -12.08 (20) | -12.63 (18) |
| 112 days (T6) | Increase | 13.58 (23) | 61.91 (23) | 30.89 (27) |
| | Decrease | -14.14 (25) | -14.85 (25) | -7.55 (20) |
Of the 230 biosynthesis pathways detected, after 14 days (T3), 93 and 131 had increased and decreased abundances by means of 19.5% and − 8.55%, respectively, in the N-only treatment, while 88 and 136 had increased and decreased abundances by means of 260% and − 17.1%, respectively, in the N + C2 treatment (Table 2; Online Resource 2, Fig. S3). After 112 days, similar numbers of pathways had increased and decreased abundances, but the magnitude of abundance differences was reduced. For example, 77 and 147 pathways had increased and decreased abundances by means of 35.4% and − 14.5%, respectively, after 112 days in N + C2.
The proportions of biosynthesis pathways with increased or decreased abundances varied among different pathway categories. For example, after 14 days (T3) in the N + C2 treatment, 13 and 23 of 35 amino acid biosynthesis pathways had increased and decreased abundances by means of 44.4% and − 15.6%, respectively. Similarly, 41 and 30 of 71 cofactor/prosthetic group/electron carrier/vitamin biosynthesis pathways had increased and decreased abundances by means of 34% and − 18.7%, respectively, after 14 days (T3) in the N + C2 treatment. In contrast, just three nucleoside and nucleotide biosynthesis pathways had increased abundances whereas 27 had decreased abundances, after 14 days (T3) in the N + C2 treatment, by means of 22.8% and − 13.2%, respectively. The largest abundance increases were observed for the cell structure biosynthesis pathway category, followed by fatty acid and lipid biosynthesis pathway category, among which 10 and eight pathways increased by means of 1,630% and 496%, respectively, after 14 days (T3) in the N + C2 treatment. In comparison, eight pathways from each of these categories increased by only 13.1% and 5.61% at the same time point in the N-only treatment. Among carbohydrate biosynthesis pathways, sucrose biosynthesis I (from photosynthesis) and sucrose biosynthesis III pathways had slightly increased abundances (6.1% and 8.5%, respectively) after 14 days (T3) in the N-only treatment, but reduced abundances (by -2.9% and − 18.9%, respectively) at the same time point in the N + C2 treatment.
Most of the 152 degradation/utilisation/assimilation pathways detected temporarily increased in abundance in each treatment (Table 2; Online Resource 2, Fig. S4). For example, after 14 days (T3), 106 and 108 pathways had increased abundances, by means of 28.2% and 527% in the N-only and N + C2 treatments, respectively. In contrast, 40 and 39 pathways had decreased abundances after 14 days (T3) in these treatments, by means of -17.6% and − 19.2%, respectively. As for the biosynthesis pathways, similar numbers of degradation/utilisation/assimilation pathways had increased and decreased abundances after 112 days, but with reduced magnitude of differences. Among the different categories of degradation/utilisation/assimilation pathways, 41 of 43 aromatic compound degradation pathways had increased abundances by a mean of 82.7% after 14 days (T3) in the N + C2 treatment, as did 13 of 14 secondary metabolite degradation pathways, by a mean of 287%, 12 of 13 amino acid degradation pathways, by a mean of 2,486%, and nine of 13 carbohydrate degradation pathways, by a mean of 112%. In contrast, 10 of 12 C1 compound utilisation/assimilation pathways decreased in abundance, by a mean of -23% after 14 days (T3) in the N + C2 treatment, while just two increased in abundance, by a mean of 266%. The carbohydrate degradation pathways included sucrose degradation III (sucrose invertase) and IV (sucrose phosphorylase), with increased abundances of 21.4% and 2.51%, respectively, after 14 days in the N-only treatment, compared to 261% and 150%, respectively, at the same time point in the N + C2 treatment. Conversely, the abundance of the sucrose degradation II (sucrose synthase) pathway increased by 93.7% after 14 days (T3) in the N-only treatment but declined by -23.9% at the same time point in the N + C2 treatment. Additionally, the abundances of glucose/glucose-1-phosphate degradation and lactose /galactose degradation I pathway abundances increased by 2.12% and decreased by -60%, respectively, after 14 days (T3) in the N-only treatment, but increased by 152% and 334%, respectively, at the same time point in the N + C2 treatment. Similarly, the abundance of the oxidative glucose degradation pathway (secondary metabolite degradation) increased by 52.8% after 14 days (T3) in the N-only treatment, compared to 1,047% and 2,486%, respectively, at the same time point in the N + C1 and N + C2 treatments.
Among the 48 precursor metabolite/energy generation pathways, after 14 days (T3), 28 and 20 pathways had increased and decreased abundances, by means of 14.2% and − 18.3%, respectively, in the N-only treatment, while 29 and 18 pathways had increased and decreased abundances by means of 78.6% and − 12.6%, respectively, in the N + C2 treatment (Table 2; Online Resource 2, Fig. S5). Again, after 112 days, the number of precursor metabolite/energy generation pathways with increased and decreased abundances was similar to at T3, but the magnitude of differences was reduced. Fifteen of 25 fermentation pathways had increased abundances, by a mean of 94.6%, after 14 days (T3) in the N + C2 treatment, compared with 14 fermentation pathways increasing by a mean of 17.4%, in the N-only treatment. Photosynthesis and glyoxylate cycle pathway abundances both increased in all three treatments, by 0.309% and 2.82%, respectively, in the N-only treatment, to 22% and 43.8%, respectively, in the N + C2 treatment, after 14 days (T3). Similarly, three of four glycolysis pathways and five of seven TCA cycle pathways had increased abundances in all three treatments, by means of 69.4% and 54.7%, respectively, after 14 days (T3) in the N + C2 treatment, compared to 7.11% and 11%, respectively, in the N only treatment.
The individual pathways with the largest abundance increases from T0 in any treatment included chitin derivatives degradation; the superpathway of L-arginine, putrescine, and 4-aminobutanoate degradation; the superpathway of L-arginine and L-ornithine degradation; enterobacterial common antigen biosynthesis; and the superpathway of lipopolysaccharide biosynthesis; variously by > 11,900% to > 23,000% after seven, 14 and/or 28 days (T2, T3 and/or T4), all in the N + C2 treatment. In addition, when both absolute pathway abundances and the magnitude of their changes were considered, those with the largest increases also included the superpathway of (Kdo)2-lipid A biosynthesis; creatinine degradation I; and polymyxin resistance (biosynthesis); variously by > 1,100% to > 3,200% after 7, 14 and/or 28 days (T2, T3 and/or T4), also all in the N + C2 treatment. Conversely, 100% declines in abundance were observed for seven biosynthesis pathways (archaetidylserine and archaetidylethanolamine biosynthesis; chorismate biosynthesis II (archaea); fosfomycin biosynthesis; streptomycin biosynthesis; the superpathway of L-tryptophan biosynthesis; the superpathway of lipopolysaccharide biosynthesis; and tetrahydromethanopterin biosynthesis), two degradation/utilization/assimilation pathways (3-hydroxypropanoate/4-hydroxybutanate cycle; L-rhamnose degradation II), and one precursor metabolite and energy generation pathway (benzoate fermentation), at a total of 38 different combinations of time point and treatment. In addition, when both pathway abundance and the magnitude of changes were considered, pathways with the largest abundance declines included aerobic respiration 1 (cytochrome c), by -5.5% to -16.4% from 14 to 56 days in the N + C1 treatment and from seven to 56 days in the N + C2 treatment; cis-vaccinate biosynthesis and gondoate biosynthesis (anaerobic), by -15.8% and − 16%, respectively, both after 28 days (T4) in the N + C2 treatment; and L-isoleucine biosynthesis IV, by -33.3% after 56 days, also in the N + C2 treatment.
Effects of urine and carbon inputs on fungal trophic modes and growth forms
Trophic modes and growth forms were assigned to 1,685 of 3,550 fungal ASVs (47%) from the FungalTraits database. The most abundant trophic modes detected were saprotrophs (67.6% of total FungalTrait-matched ASV abundance), parasites (19.6%), pathogens (9.33%), mycorrhizae (6.87%), and endophytes (1.26%) (Fig. 6a). The mean proportion of mycorrhizae dropped sharply from initial values (16.1 to 21.6%) to one day after urine application (T1) (4.4 to 8.4%) and subsequent time points in all three treatments, reaching particularly small proportions (≤ 1.3%) from 14 to 112 days in the N + C2 treatment. Saprotrophs (49.4 to 59.7%) and parasites (26.1 to 40.9%) together represented the largest mean proportions of trophic modes at all time points in the N only treatment, except for after 112 days (T6) when the mean proportion of pathogens (45.9%) exceeded those of both saprotrophs (25.6%) and parasites (18.9%). In the N + C1 and N + C2 treatments, trophic mode composition was broadly similar to that of the N only treatment until one day after urine application (T0 to T1), but became highly dominated by saprotrophs (77.9 to 94.7%) from seven days (T2) (N + C1) or 14 days (T3) (N + C2) through to 112 days (T6).
The most common growth forms detected were mycelium (68%), followed by yeasts (16.9%), zoosporic (14.8%) and dimorphic (5.1%) fungi (Fig. 6b). Mycelial ASVs accounted for the largest mean proportions of ASVs at all time points in all three treatments (except for 42% after 14 days (T3) in the N + C2 treatment). The largest mean proportions of mycelium ASVs were detected after 112 days (T6) in the N only treatment (84%), but after seven days in both the N + C1 treatment (91.8%) and the N + C2 treatment (78.9%). The mean proportions of zoosporic ASVs were between 12.6% after 112 days (T6) and 34.3% after one day (T1) in the N only treatment, with broadly similar values (19.6–38.9%) before seven days (T0-T1) in the N + C treatments. In contrast, the mean proportions of zoosporic ASVs declined sharply to ≤ 2% from 14 to 112 days in the N + C1 treatment and from seven to 112 days in the N + C2 treatment, and were largely replaced by yeasts. In N + C1, the initial mean proportion of yeast ASVs was 25.1%, declining to 2.4% after seven days (T2), before increasing to a maximal mean proportion of 37.6% after 28 days (T4), and remaining over 16.7% until 112 days (T6). In the N + C2 treatment, the mean proportion of yeast ASVs yeasts gradually increased from an initial value of 4.9% (T0) to 12.2% after seven days (T2), before sharply increasing to a maximal mean proportion of 55.6% after 14 days (T3) and remaining over 20.9% until 112 days (T6). The mean proportions of dimorphic ASVs were ≤ 3.1% at all time points in the N only treatment, but reached maximal values of 14.4% and 21.1% after 112 days in the N + C1 and N + C2 treatments, respectively.