Differences in the physicochemical properties of sediments at different sites
The physical and chemical properties of sediments in the Jialing River are affected by many factors, such as water and soil loss in the riparian zone, pollution discharge, and river disturbance. Therefore, the physical and chemical properties of sampling points in different river sections exhibited certain differences. Table S1 shows that the pH of the sediments at site B was higher than that at the other sites, indicating that NPSP significantly reduced the pH of the sediments (P<0.05). Compared with that in B, the AN content in A and C was significantly increased (P<0.05). The TC content varied from 5.72 to 15.37 g·kg−1, the TN content varied from 0.36 to 1.02 g·kg−1, and the TOC content varied from 4.68 to 12.11 g· kg−1. These 3 physicochemical parameters showed the same change trends at the different sites. There were certain differences in the TP content and C/N value at different sites, but there was no obvious change trend. Hence, the pH and AN content of the sediments were significantly affected by NPSP, while the other physicochemical properties of the sediments had no significant relationship with NPSP.
DNA sequencing data and ASV distribution of fungi in sediment samples from different sites
A total of 1 294 031 optimized sequences were detected in 18 sediment samples through high-throughput sequencing, with a total base number of 284 476 801 bp, an average sequence number of 71 891, an average base number of 15 804 267 bp, and an average base length of 219.9 bp. The sequences were analysed by ASV taxonomy according to 100% similarity, and 11 184 ASVs were obtained. As shown in Fig. 2, the distribution numbers of ASVs at the 6 sampling sites decreased in the order A1 > C1 > C2 > B1 > A2 > B2, and the numbers of ASVs detected were 4 003, 2 297, 3 922, 2 216, 1 617, and 2 602, respectively.
The numbers of ASVs of the sediment fungal communities from sites A and C of the same estuary were similar (Fig. 2a, 2b). The number of shared ASVs between A and C (1372 shared by A1 and C1 and 773 shared by A2 and C3) is shown in the Venn diagram. A small number of ASVs shared between B and other sites (671 shared by A1 and B1, 624 shared by B1 and C1, 360 shared by A2 and B2, and 352 shared by B2 and C2) are also shown. This shows that the distribution of sediment fungal ASVs in the main stream of the Jialing River is significantly affected by the NPSP tributary and is affected by other environmental factors.
As shown in Fig. 2 (d, e, f), the number of ASVs shared between each pair of sites (749 shared by A1 and A2, 474 shared by B1 and B2, and 825 shared by C1 and C2) and unique to each site (3 254 unique to A1, 1 467 to A2, 1 823 to B1, 1 143 to B2, 3 097 to C1, and 1 777 to C2) are shown in the Venn diagram (Fig. 2d, 2e, 2f). The fungal communities of the two NPSP tributaries exhibited certain differences, and the distribution of fungal ASVs in the sediments of the main stream of the Jialing River showed changes.
Diversity analysis of the fungal community in sediments from different sites
α-Diversity analysis of the fungal community in sediments from different sites
Several α-diversity indexes were calculated to describe the richness and diversity of sediment fungal ASVs at the different sites (Table 1). The independent t-tests used to explore the differences between the six sites indicated that the richness of the sediment fungal community in C1 was significantly higher than that in other sites (P < 0.05). This result indicates that NPSP increased the diversity of fungi in the Jialing River sediments. Similarly, the richness of the sediment fungal community in C2 was significantly higher than that in A2 and B2 (P < 0.05). It was further verified that NPSP significantly increased the fungal diversity of sediments (P<0.05).
Table 1
Comparison of the α-diversity index of sediment fungi at different sampling sites. Different lowercase letters denote significant differences between sampling sites at P < 0.05. The maximum values in each column are denoted by “a”. α-Diversity indexes were calculated at the ASV level.
site | Ace | Chao | Shannon-Wiener | Simpson |
A1 | 1717.33±283.83a | 1717.33±283.83a | 6.23±0.28ab | 0.0100±0.0073b |
B1 | 1033.67±71.23bc | 1033.67±71.23bc | 5.25±0.13bc | 0.0218±0.0075b |
C1 | 1753.33±77.36a | 1753.33±77.36a | 6.38±0.08a | 0.0051±0.0008b |
A2 | 985.33±214.76bc | 985.33±214.76bc | 3.93±1.33d | 0.1874±0.1833a |
B2 | 747.33±66.29c | 747.33±66.29c | 4.50±0.17cd | 0.0431±0.0015b |
C2 | 1209.00±46.87b | 1209.00±46.87b | 4.44±0.36cd | 0.1029±0.0240ab |
β-Diversity analysis of the fungal community in sediments from different sites
The correlations of and differences in the sediment fungal communities among the six sampling sites were compared by PCA of the Pearson distance algorithm at the species level (Fig. 3). The cumulative explained variance of the first axis and the second axis reached 83.64%, 92.01%, and 61.97%. The fungal communities at different sampling sites in the same estuary showed no obvious overlap with each other and could be separated from each other. Analysis of similarities (ANOSIM) of the groups showed that the fungal communities in sites A and C of the same estuary shared high similarity and were significantly different from the fungal communities in B (P<0.05). In addition, Fig. 3c shows that the fungal communities of B2 and Dong River Estuary sediments shared a certain similarity, and the fungal community diversity of C2 exhibited higher variation, which was affected by NPSP in the Xichong River (P<0.05).
Community structure and indicator species of fungi in sediments from different sites
Fungal community structure characteristics of sediments from different sites at the phylum level
Taxonomic analysis of ASVs at the phylum level (Fig. S1) showed that the sediment fungi from all the sites belonged to 14 known phyla. Among them, Rozellomycota, Ascomycota, Chytridiomycota, Basidiomycota, Mortierellomycota and Zoopagomycota were the main fungal phyla at each sampling site (relative abundance> 1%). Olpidiomucota, Kickxellomycota, Glomeromycota, Blastocladiomycota, Calcarisporiellomycota, Mucoromycota, Basidiobolomycota, and Monoblepharomycota were rare fungal phyla at each sampling site (relative abundance< 1%).
Fungal community structure characteristics of sediments from different sites at the genus level
Taxonomic analysis of ASVs at the genus level (Fig. S2) showed that the sediment fungi from all the sites belonged to 778 genera. Among them, Cladosporium, Paraphaeosphaeria, Saitozyma, Pseudeurotium, Trichoderma, Epicoccum, Penicillium, Plectosphaerella, Didymella, Talaromyces, Tausonia, Botrytis, Acaulopage, and Podospora were known dominant fungal genera at each site. Most of the fungi belonged to rare genera or genera that could not be classified and named.
Analysis of indicator fungi in sediments from different sites
The LEfSe analysis results show that the characteristics of the fungal communities in the sediments differed among sites (Fig. 4). At the phylum level, Chytridiomycota was the dominant fungal phylum with significant differences in B, while Rozellomycota was the dominant fungal phylum with significant differences in C. At the class level, Dothideomycetes in B and Saccharomycetes in C were the dominant fungi with significant differences. At the order level, Capnodiales, Glomerellales, Xylariales, and Chaetothyriales in the sediments of B were the dominant fungi with significant differences. Pleosporales, Morosphaeiaceae, Trichosphaeriales, Trichosporonales, and GS11 in the sediments of A were the dominant fungi with significant differences. Microascales, Saccharomycetales, Branch02, and Branch03 in the sediments of C were the dominant fungi with significant differences. At the family level, Mycosphaerellaceae, Cladosporiaceae, Phaeosphaeriaceae, Periconiaceae, Plectosphaerellaceae, Nectriaceae, Apiosporaceae, Rhynchogastremataceae were the dominant fungal families with significant differences in the sediments of B. Extremaceae, Morosphaeiaceae, Dictyosporiaceae, Hypocreaceae, Trichosphaeriaceae, and Trichosporonaceae were the dominant fungi families with significant differences in the sediments of A. Extremaceae and Inocybaceae were the dominant fungal families with significant differences in the sediments of C. At the genus level, Cercospora, Cladosporium, Dokmaia, Setophaeosphaeria, Paraphoma, Neosetophoma, Periconia, Plectosphaerella, Claviceps, Botrytis, and Papiliotrema were the dominant fungal genera with significant differences in the sediments of B. Acrocalymma, Emericellopsis, Trichoderma, Podospora, Ciboria, and Apiotrichum were the dominant fungal genera with significant differences in the sediments of A. Exosporium, Phialosimplex, Candida, Inocybe, Tausonia, and Slooffia were the dominant genera with significant differences in the sediments of C. At the species level, Setophaeosphaeria badalingensis, Periconia byssoides, Aureobasidium leucospemi, Claviceps sorghicola, Papiliotrema flavescens, and Chytridiomycota sp. were the dominant species with significant differences in the sediments of B. Podospora communis, Talaromyces helices, Basidiomycota sp., and GS11 sp. were the dominant species with significant differences in the sediments of A. Exosporium sp., Acaulium sp., Phialosimplex sp., Exophiala oligosperma, Candida sake, Tausonia sp., Slooffia cresolica, and Branch02_sp were the dominant species with significant differences in the sediments of C. In addition, there were some unclassified and unnamed fungi in the sediments of various sites.
Fungal functional groups of sediments in different sites
The FUNGuild microecological analysis tools were used to predict the utilization of similar environmental resources based on fungal communities. As shown in Fig. S3, the detected sediment fungi were classified into three types (symbiotrophs, pathotrophs, and saprotrophs) according to their absorption and utilization of environmental resources. The fungi were classified into 12 functional groups, including litter saprotrophs, soil saprotrophs, wood saprotrophs, bryophyte parasites, lichen parasites, rhododendron ericoid mycorrhizae, ectomycorrhizae, animal pathogens, dung saprotrophs, plant pathogens, endophytes, and fungal parasites. The 12 functional types were very evenly distributed among various plots, and NPSP did not have a significant impact on the different environmental resource utilization mechanisms.
PICRUSt 2 software was used to predict and analyse the functional metabolic pathways of sediment fungal communities (Table 2). All the samples contained a total of 75 metabolic circulation pathways. Among them, 25 main metabolic circulation pathways showed significant differences among different types of sites (relative abundance of functional gene sequences>1%). The levels of the metabolic circulation pathways PWY-7288, PWY66-409, and PWY-5189 in B were significantly higher than those in the other sites (P<0.05), and the levels of GLYOXYLATE-BYPASS, VALSYN-PWY, PWY-7411, PWY66-422 in B were significantly lower than those in the other sites (P<0.05). The levels of the metabolic circulation pathways GLYOXYLATE-BYPASS, PWY-7210, PWY-6317, and PWY-7385 in A were significantly higher than those in the other sites (P<0.05). The levels of PWY-7007 and PWY-7208 in A were significantly lower than those in the other sites (P<0.05). The levels of the metabolic circulation pathways NONOXIPENT-PWY, PENTOSE-P-PWY, and PWY-6837 in C were significantly higher than those in the other sites (P<0.05). The levels of PWY-7118, PWY-5920, and PWY-6609 in the C group were significantly lower than those in the other groups (P<0.05).
Table 2
Comparison of fungal metabolic pathways in the sediments at the sampling sites. Different lowercase letters denote significant differences between sampling sites at P < 0.05. The maximum values in each column are denoted by “a”.
metacyc pathway | description | A | B | C |
PWY-7288 | fatty acid | 0.0337±0.0037c | 0.0412±0.0014a | 0.0337±0.0016b |
GLYOXYLATE-BYPASS | glyoxylate cycle | 0.0272±0.0014a | 0.0243±0.0005c | 0.0260±0.0004b |
PWY-7184 | pyrimidine deoxyribonucleotides de novo biosynthesis I | 0.0214±0.0003b | 0.0236±0.0009a | 0.0223±0.0010ab |
PWY-7111 | pyruvate fermentation to isobutanol | 0.0206±0.0004ab | 0.0202±0.0002b | 0.0206±0.0003a |
PWY-7228 | superpathway of guanosine nucleotides de novo biosynthesis I | 0.0192±0.0010b | 0.0203±0.0004a | 0.0194±0.0007ab |
NONOXIPENT-PWY | pentose phosphate pathway | 0.0183±0.0011b | 0.0182±0.0004b | 0.0206±0.0026a |
PWY-7007 | methyl ketone biosynthesis | 0.0164±0.0040b | 0.0209±0.0018a | 0.0196±0.0003a |
PWY-7197 | pyrimidine deoxyribonucleotide phosphorylation | 0.0183±0.0013b | 0.0199±0.0006a | 0.0188±0.0006ab |
PWY-7208 | superpathway of pyrimidine nucleobases salvage | 0.0178±0.0007b | 0.0185±0.0003a | 0.0185±0.0004a |
VALSYN-PWY | L-valine biosynthesis | 0.0180±0.0007a | 0.0177±0.0002b | 0.0185±0.0001a |
PWY-5067 | glycogen biosynthesis II | 0.0174±0.0011b | 0.0183±0.0001a | 0.0171±0.0007ab |
PANTO-PWY | phosphopantothenate biosynthesis I | 0.0168±0.0006ab | 0.0164±0.0002b | 0.0171±0.0004a |
PENTOSE-P-PWY | pentose phosphate pathway | 0.0161±0.0003b | 0.0161±0.0002b | 0.0168±0.0008a |
PWY-7118 | chitin degradation to ethanol | 0.0169±0.0021a | 0.0171±0.0011a | 0.0139±0.0004b |
PWY-7411 | superpathway of phosphatidate biosynthesis | 0.0159±0.0005a | 0.0151±0.0004b | 0.0161±0.0006a |
PWY-6837 | fatty acid beta-oxidation V | 0.0131±0.0025b | 0.0150±0.0009b | 0.0175±0.0021a |
PWY-7282 | 4-amino-2-methyl-5-phosphomethylpyrimidine biosynthesis | 0.0157±0.0008a | 0.0150±0.0006ab | 0.0147±0.0001b |
PWY66-422 | D-galactose degradation V | 0.0151±0.0004a | 0.0145±0.0004b | 0.0151±0.0003a |
PWY-7210 | pyrimidine deoxyribonucleotides biosynthesis from CTP | 0.0156±0.0018a | 0.0138±0.0011b | 0.0137±0.0012b |
PWY-5920 | superpathway of heme biosynthesis from glycine | 0.0134±0.0007a | 0.0120±0.0009a | 0.0128±0.0002b |
PWY66-409 | superpathway of purine nucleotide salvage | 0.0115±0.0020b | 0.0140±0.0009a | 0.0119±0.0010b |
PWY-5189 | tetrapyrrole biosynthesis II | 0.0125±0.0010b | 0.0103±0.0011a | 0.0117±0.0001b |
PWY-6609 | adenine and adenosine salvage III | 0.0120±0.0016a | 0.0123±0.0004a | 0.0097±0.0015b |
PWY-6317 | galactose degradation I | 0.0122±0.0016a | 0.0100±0.0007b | 0.0104±0.0015b |
PWY-7385 | 1,3-propanediol biosynthesis | 0.0119±0.0013a | 0.0099±0.0009b | 0.0104±0.0008b |
Relationship between the physicochemical properties of sediments and fungal community diversity
The correlation analysis between the sediment physicochemical properties and sediment fungal α-diversity showed (Table S2) that the Ace index and Chao index were significantly negatively correlated with sediment pH (P<0.05) and significantly positively correlated with the sediment TN and AN levels (P<0.05). This indicates that the pH, TN and AN levels have significant effects on the fungal species richness of sediments.
Redundant analysis of sediment fungal community structure and sediment physical and chemical properties at the species level (Fig. 5). The results show that the cumulative explained variation of the two axes reached 75.95%, reflecting more than 70% of the sediment fungal community change characteristics and influencing factors. The results of the displacement test showed that the sediment pH (r2=0.6189, P=0.002), TP content (r2=0.4486, P=0.016) and AN content (r2=0.3321, P=0.049) were the key environmental factors that led to structural variation in the fungal communities.