Habitat information and soil nutrient characteristics
A habitat survey of three wild Paphiopedilum species revealed that (Table 1) P. armeniacum grows in mountain shrubs at an altitude of approximately 1700-2000 m, and the slope is usually 30-55°. P. emersonii is distributed in the evergreen and deciduous broad-leaved mixed forest at an altitude of approximately 300-800 m in karst low-mountain hills, with a very steep growth slope (75-90°). P. wenshanense is distributed in shrubs on normal landforms and karst landforms, with altitudes of approximately 1500-1600 m. The three species have similar habitats. They all like to grow on shady slopes such as those in the north and northwest and like to grow on negative terrain such as tree roots, stone pits and stone crevices. The soil nutrients of the three Paphiopedilum species were analyzed and tested by one-way analysis of variance (Table 2). The results showed that there was no significant difference in total nitrogen, total phosphorus, total potassium, organic matter, available potassium or pH among the three species. The content of alkali-hydrolyzable nitrogen in the soil of P. emersonii was significantly greater than that in the soil of P. wenshanense. The available phosphorus in the soil of P. emersonii and P. armeniacum was significantly greater than that in the soil of P. wenshanense.
Table 1 Habitat information of three Paphiopedilum species
Specie
|
Altitude(m)
|
Geomorphology
|
Vegetational Form
|
Slope(°)
|
Aspect
|
Microhabitat
|
P. armeniacum
|
1776-2020
|
Normal Geomorphology
|
Shrubwood
|
30-55
|
North‒West、North
|
Tree Root、Swallet
|
P. emersonii
|
389-835
|
Karst
|
Evergreen And Deciduous Broad Leaved Forest
|
75-90
|
North South East West
|
Stonewall
|
P. wenshanense
|
1525-1630
|
Karst,Normal Geomorphology
|
Shrubwood
|
30-50
|
Northwest
|
Tree Roots, Stone Crevices
|
Table 2 Differences in the soil nutrients in the habitats of the three paphiopedilum species
|
P.armeniacum
|
P.wenshanense
|
P.emersonii
|
TN
|
5.19±1.28a
|
1.81±1.58a
|
7.88±5.06a
|
TP
|
0.62±0.24a
|
0.35±0.13a
|
0.57±0.13a
|
TK
|
5.18±2.23a
|
4.66±1.08a
|
3.19±0.76a
|
SOC
|
128.49±31.11a
|
43.18±37.63a
|
150.70±97.24a
|
AN
|
223.58±42.92ab
|
102.95±80.55b
|
316.74±85.20a
|
AP
|
4.1±2.19a
|
1.08±0.08b
|
5.04±1.30a
|
AK
|
138.67±45.24a
|
194.33±122.15a
|
70±41.04a
|
pH
|
6.76±1.046a
|
7.27±0.62a
|
7.77±0.32a
|
Note: The values are the means ± standard errors. Different lowercase letters in the same row indicate significant differences between horizontal gradients (P < 0.05). TN: total nitrogen; TP: total phosphorus; TK: total potassium; TOC: total organic carbon; AN: alkali-hydrolyzable nitrogen; AP: available phosphorus; AK: available potassium
Species dilution curve and Venn diagram
The dilution curve can truly reflect the sequencing depth of the sample sequence. As shown in Fig. 2a, at a similarity of 97%, the soil fungal dilution curves of the three Paphiopedilum species tended to decrease, indicating that the sample size could represent the soil fungal community in the plant habitat as a whole. A total of 2,161,515 pairs of reads were obtained from 27 fungal habitat samples. After quality control and splicing of the double-ended reads, a total of 2,154,184 clean reads were generated. Each sample produced at least 79,308 clean reads, with an average of 79,785 clean reads. High-throughput sequencing analysis was performed based on the 97% similarity tag classification as an OTU standard, and a total of 1068 operable units were obtained. Among them, 452 unique OTUs were found in the soil of P. emersonii (Fig. 2b), followed by P. wenshanense (n=232) and P. armeniacum (n=211). There were 65 OTUs in the P. wenshanense and P. armeniacum habitat soils, 25 OTUs in the P. armeniacum and P. emersonii habitat soils, and 46 OTUs in the P. emersonii and P. wenshanense habitat soils; moreover, there were 37 common OTUs in the three Paphiopedilum habitat soils.
According to the species annotation (Table 3), a total of 336 fungal species belonging to 11 phyla, 30 classes, 74 orders, 157 families, and 272 genera were identified. A total of 230 species of fungi belonging to 10 phyla, 26 classes, 62 orders, 127 families, and 202 genera were identified in the soil of P. emersonii. A total of 138 species of fungi belonging to 9 phyla, 21 classes, 44 orders, 86 families, and 116 genera were identified in the soil of P. armeniacum. A total of 145 species of fungi belonging to 7 phyla, 21 classes, 52 orders, 98 families, and 126 genera were identified in the soil of P. wenshanense.
Table 3 Number of fungal species in the soil of three Paphiopedilum species
Species
|
Phylum
|
Class
|
Order
|
family
|
Genus
|
Species
|
OTUS
|
P.emersonii
|
10
|
26
|
62
|
127
|
202
|
230
|
560
|
P.armeniacum
|
9
|
21
|
44
|
86
|
116
|
138
|
338
|
P.wenshanense
|
7
|
21
|
52
|
98
|
126
|
145
|
380
|
Total
|
11
|
30
|
74
|
157
|
272
|
336
|
1068
|
Fungi composition and functional group composition in the habitat soil of three paphiopedilum species
The relative abundance of fungal groups in the habitat soil of the three Paphiopedilum species at the phylum and genus levels is shown in Fig. 3. The dominant group of fungi in the soil of P. wenshanense was Calcarisporiellomycota, and no obvious dominant group was found in the soil of P. armeniacum. There were dominant fungal groups, such as Kickxellomycota, Entorrhizomycota, Olpidiomycota and Rozellomycota, in the P. emersonii habitat. At the genus level, there were unclassified_Sordariomycetes, Sebacina, unclassified_Basidiomycota, Boletus, unclassified_Boletaceae, Archaeorhizomyces and other dominant fungi in the habitat soil of P. wenshanense. Unclassified _ Thelephoraceae, Hygrocybe, unclassified_Serendipitaceae, unclassified_Ascomycota, Tomentella and unclassified_Agaricomycetes were found in the soil fungal habitat of P. armeniacum. Acremonium, unclassified_Fungi, unclassified_Chaetothyriales, Apodus, unidentified, unclassified_Hypocreales and other soil fungi were found in the soil of P. emersonii.
Based on the ecological role of fungi in the environment, the functional classification and annotation of soil fungi in the soil of three Paphiopedilum species were carried out by using the FUNGuild microecological tool. The functions of soil fungi can be divided into three types according to the nutritional mode: saprophytic nutrition, symbiotic nutrition and pathological nutrition. In the soil of the three Paphiopedilum species, the saprophytic and symbiotic nutrient types were dominant (Fig. 4a). In particular, the relative abundance of the two nutritional fungi in the soil of P. armeniacum accounted for 98%. It also accounts for more than 80% of the habitat of P. wenshanense and P. emersonii. The fungal functional groups were further divided into 10 categories by environmental resource absorption (Fig. 4b). These included Undefined Saprotroph, Ectomycorrhizal, Undefined-Biotroph, Soil Saprotroph, Fungal Parasite, Wood Saprotroph, Plant Saprotroph, Animal Pathogen, Plant Pathogen and Orchid Mycorrhizal. Among them, undefined saprophytic fungi and ectomycorrhizal fungi account for a large proportion of the three Paphiopedilum species in the soil and are two types of fungal functional groups that play important ecological roles. Undefined saprophytic fungi, ectomycorrhizal fungi, and undefined trophic fungi were dominant in the habitat soil fungal functional group of P. armeniacum, accounting for more than 95% of the relative abundance, and some orchid mycorrhizal and animal pathogenic fungi. The main fungal functional groups of P. wenshanense in the soil habitat were saprophytic fungi, animal pathogens, plant parasitic fungi, plant saprophytic fungi, wood saprophytic fungi, plant pathogens and other dominant functional groups. The soil fungal functional groups of P. emersonii were also rich, and their relative abundances were relatively similar.
Diversity analysis
The alpha diversity analysis of the habitat soil fungal communities of the three Paphiopedilum species revealed no significant differences in the ACE and Chao1 indices, indicating that there was no significant difference in the community abundance of the habitat soil fungi among the three species of Paphiopedilum. The Simpson index and Shannon index of P. emersonii were significantly greater than those of P. armeniacum and significantly greater than those of P. wenshanense(Fig 5). In addition, there was no significant difference in the four diversity indices between P. armeniacum and P. wenshanense.
To clarify the overall differences in the soil fungal community structure among the three Paphiopedilum species, the beta diversity was analyzed via nonmetric multidimensional scaling (NMDS) based on the Bray‒Curtis distance (based on fungal abundance and species presence or absence). Prior to this, to verify the reliability of species as a grouping unit, we used permutational MANOVA. The results showed that the differences in the habitat soil fungal community structure among the three Paphiopedilum species were significantly greater than the intraspecific differences, indicating that the grouping results were reliable (Figure 6). The R values were 0.214 and 0.388 at the phylum and genus levels, respectively, indicating that the grouping method explained 21.4% and 38.8% of the sample differences, respectively.
Figure 7 shows the results of the NMDS analysis, and the ordination axis was set to 2. The stress values (strees) of the soil fungal community in the soil of the three Paphiopedilum species at the phylum and genus classification levels were less than 0.2, indicating that the results have explanatory significance. The stress values at the phylum and genus levels were 0.0071 and 0.159(Fig. 7), respectively, indicating that the differences in the habitat soil fungal community structure of the three Paphiopedilum species were more obvious at the phylum level.
Soil fungal co-occurrence network of three Paphiopedilum species
To study the potential interactions between the soil fungi in the three Paphiopedilum species and the changes in the co-occurrence network, an OTU-level co-occurrence network of the soil fungi of the three Paphiopedilum species was constructed based on random matrix theory. The same threshold (r > 0.6, p < 0.01) was used to construct the co-occurrence network, and the changes in the co-occurrence network were compared and analyzed. As shown in Fig. 8, there were 74 nodes and 606 edges in the habitat soil fungal co-occurrence network of P. emersonii, of which 93.56% were positively correlated and only 6.44% were negatively correlated(Fig. 8A). There were 77 nodes and 479 edges in the co-occurrence network of soil fungi in the habitat of P. armeniacum. The proportion of positively correlated edges was 70.56%, and the proportion of negatively correlated edges was 29.44%(Fig. 8B). The aggregation of the soil fungal co-occurrence network in the P. wenshanense habitat was the smallest, with only 53 nodes and 183 nodes. The proportion of positive correlation edges was 87.43%, and the proportion of negative correlation edges was 12.57%(Fig. 8C). This study revealed that the co-occurrence network of the soil fungi of P. emersonii and P. armeniacum had a high degree of modularity and a large proportion of positive effects, indicating that the fungal co-occurrence network included modules that resisted changes in the external environment. This symbiotic model may help maintain community structure to resist adverse environmental conditions and contribute to the effective degradation of organic matter.
Relationships between habitat soil fungi and soil nutrients of three paphiopedilum species
The correlation between soil nutrients and the effect on fungal alpha diversity was analyzed by the Mantel test. The results showed that (Fig. 9A) there were some significant correlations between the soil nutrient factors. The soil total nitrogen content was significantly positively correlated with organic carbon, alkali-hydrolyzable nitrogen and available phosphorus, and total phosphorus was significantly positively correlated with available phosphorus. Available potassium was significantly negatively correlated with total phosphorus, alkali-hydrolyzable nitrogen and available phosphorus, and the pH was significantly negatively correlated with total potassium. The soil pH significantly affected the Shannon index and Simpson index of the soil fungi. Redundancy analysis was performed with soil nutrients using the fungal groups with the ten most dominant fungal taxa as response variables. The results showed that the first axis (Fig 9B) explained 60.41% of the variance. The second axis explained 20.29% of the variance. A total of 80.70% of the changes in the horizontal direction of the fungal dominant groups were explained, of which total nitrogen, organic carbon, and alkali-hydrolyzed nitrogen were the most important factors. Further Spearman correlation analysis was used to reveal the associations between soil nutrients and dominant fungal groups (Fig. 10). In the P. emersonii habitat, total potassium was significantly negatively correlated with Rozellomycota, Mortierellomycota and Ascomycota and significantly positively correlated with Basidiomycota. Alkaline nitrogen was significantly negatively correlated with unclassified fungi and Chytridiomycota. The pH was significantly positively correlated with unclassified fungi and Chytridiomycota. In the P. armeniacum, Rozellomycota, Basidiomycota, and Ascomycota habitats, all nutrient factors except total potassium were significantly correlated. The abundance of Rozellomycota was negatively correlated with available potassium and positively correlated with available potassium. Basidiomycota were positively correlated with available potassium and negatively correlated with the other factors. The abundance of Ascomycota was also negatively correlated with available potassium and positively correlated with the other phyla. There was a negative correlation between unclassified fungi and total potassium. In the P. wenshanense habitat, unclassified fungi, Basidiomycota, Ascomycota and Chytridiomycota were significantly correlated with total nitrogen, total phosphorus, organic carbon, alkali-hydrolyzed nitrogen, available phosphorus and available potassium but were not significantly correlated with the remaining factors. The abundance of Mortierellomycota was significantly negatively correlated with organic carbon and available potassium, and the abundance of Glomeromycota was significantly negatively correlated with pH.