Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China

Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China Li Tian, Mingtai An, Feng Liu, Yang Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4245399/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Paphiopedilum armeniacum , Paphiopedilum wenshanense and Paphiopedilum emersonii are critically endangered wild orchids. The population is threatened, and the number of natural distribution sites has plummeted. Ex situ conservation and artificial breeding are the keys to maintaining the population to ensure the success of ex situ conservation and field return in the future. The habitat characteristics and soil nutrient information of the last remaining wild distribution sites of the three species were studied. ITS high-throughput sequencing was used to reveal the composition and structure of the soil fungal community, analyze its diversity and functional characteristics, and reveal its relationship with soil nutrients. Results: The three species preferred to grow on low-lying, ventilated and shaded negative terrain with good water drainage. There were significant differences in soil alkali-hydrolyzed nitrogen and available phosphorus among the three species. There were 336 fungal species detected in the samples. On average, there were different dominant groups in the soil fungal communities of the three species. The functional groups of soil fungi in the habitats were dominated by saprophytic fungi and ectomycorrhizae, with significant differences in diversity and structure. The co-occurrence network of habitat soil fungi was mainly positive. Soil pH significantly affected soil fungal diversity in the habitats of the three paphiopedilum species. The study and analysis confirmed that the dominant groups of soil fungi were significantly correlated with soil nutrients. Conclusion: The three species have similar habitat preferences, but there are significant differences in soil fungal composition, community structure and diversity. The functional group of fungi contains abundant saprophytic fungi, ectomycorrhizae, and a small amount of orchid mycorrhizae. The symbiotic relationships of the three species of soil fungi were harmonious, which was conducive to resistance to adverse environments. Soil environmental factors were significantly correlated with soil fungal communities, and pH significantly controlled fungal diversity. Our study on the habitat characteristics and soil fungal communities of the three wild paphiopedilum species laid a foundation for future ex situ conservation and field return work. Orchid Paphiopedilum Soil fungi Habitat characteristics Ex situ conservation Figures Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Orchidaceae is one of the largest families of higher plants and one of the most evolutionarily morphologically rich plant families in nature. There are approximately 25,000-35,000 Orchidaceae species in 800 genera worldwide[1, 2]. Orchid organs are highly specialized, diverse, and highly adaptable to the environment and are widely distributed in various terrestrial ecosystems except for the poles and extreme arid desert regions[3, 4]. Orchids have highly specialized mycorrhizal communities and special pollination mechanisms [5] and are extremely sensitive to habitat changes [6, 7]. Orchids also have unique ornamental functions and medicinal value [8], making them “flagship” species in the ecological environment[9]. At present, due to severe anthropogenic activity and the impact of global climate change, wild orchids are facing great pressure to survive. All wild orchids in the world are protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and their trading and trading are explicitly prohibited [10]. Paphiopedilum belongs to the Orchidaceae family and has large, slip-shaped lips on its petals [11, 12]. As the flower shape is similar to that of the slippers of noble European women in the Middle Ages, Paphiopedilum is also known as the slipper orchid, which is the most ornamental group of orchaceae plants and enjoys a high reputation among international flower ornamentals. This topic has attracted the attention of many scientific researchers and flower lovers around the world [13]. Due to the ornamental value of Paphiopedilum , the wild population is subject to predatory mining and the smuggling trade [14]. In addition, it is difficult for this group of plants to reproduce in the wild, and the species and number of plants are rapidly reduced or even extinct [15]. At present, all Paphiopedilum species are listed on the CITES List and the IUCN Red List of endangered species, and in China, all of them are listed on the State Key Wild Plant Protection List, which is rare among the rare and endangered plant groups in the world [16]. Southwest China is the origin and differentiation center of Paphiopedilum and the distribution hotspot of Paphiopedilum . However, according to a recent survey of wild Paphiopedilum plants, the plants of Paphiopedilum in Southwest China are suffering devastating damage, especially Paphiopedilum armeniacum ( P. armeniacum ), Paphiopedilum wenshanense ( P. wenshanense ) and Paphiopedilum emersonii ( P. emersonii ), which are the most endangered[17]. At present, fewer than 10 natural distribution sites of these three Paphiopedilum species have been found, and the latter habitats are at risk of degradation and loss. These three species of Paphiopedilum are listed as small populations on the wild plant protection list in China, which means that there is a risk of extinction in the wild at any time, which also indicates the urgency and priority of protection. Understanding the ecological habits of orchids is a prerequisite for scientific conservation work, especially for ex situ conservation of endangered species [18, 19]. In the natural environment, because the seeds of orchids are small, the germ is degraded and lacks nutrients, and the germination of seeds depends on the help of fungi, which can provide necessary resources such as carbon, nitrogen and phosphorus [20]. The entire growth cycle of orchids also depends on compatible mycorrhizal fungi in the soil, whether specialized or compatible. Considering the heavy dependence of orchids on mycorrhizal fungi, it can be predicted that their spatial distribution largely depends on the spatial distribution of microorganisms in the soil of the habitat, and the composition of fungi in the soil of the habitat is a key feature of the ecological habits of orchids [21, 22]. There is evidence that changes in mycorrhizal fungal communities driven by habitat conditions can directly or indirectly affect the distribution dynamics of terrestrial orchids [23, 24]. In addition, some seed introduction experiments showed that the seeds could germinate outside the densely distributed areas of orchids, indicating that orchid seeds may recruit fungi from the soil of the habitat to complete symbiosis, germination and growth [25, 26] and gradually form a microbial community with orchid roots as the distribution core. Therefore, the soil fungal community in a habitat may be the origin of orchid mycorrhizal fungi [27-29]. Moreover, locations close to orchid plant growth represent fungal hotspots for seed germination and new plant establishment. However, most of the current studies have focused on the endophytic fungi and rhizosphere fungal communities of orchids [30-33] while ignoring the fungal communities in habitat soil. This lack of understanding of the species source and process of orchid mycorrhizal fungal community construction restricts the development of simulated environment construction for ex situ conservation. At present, due to the reduction in the number of remaining wild populations and the degradation of habitats, the supplementation of seedlings is difficult to guarantee, and the survival of wild populations is at stake. In this regard, a necessary and urgent solution is to construct a suitable habitat for the growth of Paphiopedilum in a good artificial environment, expand its population size, and ultimately return to the wild. To ensure success, it is crucial to fully reveal the soil fungal communities in the last remaining habitat of the three Paphiopedilum species, including how they change spatially and respond to the biotic environment. In this study, P. armeniacum , P. wenshanense , P. emersonii , were used as research objects to comprehensively investigate the environmental characteristics of the remaining habitats, and soil samples were collected from the habitats of the three Paphiopedilum populations. The soil fungi in their habitats were determined based on high-throughput sequencing methods to clarify the fungal community composition structure, fungal functional groups, and fungal community diversity. The dynamic changes in fungi in the soil of Paphiopedilum habitat were revealed, and the response of the fungal community structure in the habitat to the biotic environment was initially explored, compensating for the lack of research on the habitat characteristics of the wild population of this group of species. In addition. This study can also provide a reference for ex situ conservation, artificial breeding environment selection and the construction of this group of plants. Materials and methods Sample collection In this study, after fully investigating the natural distribution of three Paphiopedilum species, three natural distribution points with good growth and no man-made damage were selected for each species (Fig. 1). P. armeniacum is distributed in Nujiang Prefecture, Yunnan Province. P. wenshanense is distributed in Wenshan Prefecture, Yunnan Province. P. emersonii is located at the junction of Guizhou Province and Guangxi Province. First, the last habitat of Paphiopedilum was observed, and information on the latitude, longitude, altitude, slope, aspect and vegetation type was recorded. Sampling was carried out at 10 cm outside the dense distribution area of Paphiopedilum species. We set the direction of possible landing of seeds as the sampling direction and extracted 20 grams of repeated soil samples in 3 directions at 5 cm from the soil layer. Soil fungal samples from 27 habitats of three species were collected. Before sampling, the air environment was disinfected with 75% medical alcohol to remove the undecomposed litter. To collect representative soil samples, the soil was transferred to a 4 °C refrigerator within 12 hours after sampling to minimize DNA degradation. For meta-barcode analysis, a small amount of soil (< 250 mg) was added to BashingBead TM Lysis Tubes (Zimo Research, Cambridge Bioscience, Cambridge, UK) to preserve environmental DNA for the extraction and amplification process. Soil nutrient test samples According to the above sampling method, 200 g of soil (at a depth of 0 ~ 10 cm) from each habitat was collected, placed in a sampling bag and returned to the laboratory. Soil nutrient analysis The soil physiochemical properties were analyzed according to Bao and are described briefly as follows[34]. The pH was determined by the water:soil = 2.5:1 extraction pH meter (PHS-3G) method. The water content was determined by the 105 °C drying–weighing method. The organic carbon content was determined by the KCr2O7-H2SO4 external heating method. Total nitrogen was determined by the semimicro Kjeldahl method; total phosphorus was determined by the HClO4-H2SO4 digestion-molybdenum antimony colorimetric method. Total potassium was determined by NaOH melting-flame spectrophotometry. Available phosphorus was determined by a 0.03 mol/L NH4F-0.025 mol/L HCl extraction-molybdenum antimony anti-colorimetric method. Available potassium was extracted by a 1 mol/L neutral NH4OAC-flame photometer. Ammonium nitrogen and nitrate nitrogen were determined by a 2 mol/L KCl extraction-continuous flow analyzer. The soil enzyme activities were determined using an enzyme analysis kit (Yangling Xinhua Ecological Technology Co., Ltd., Shanxi, China). Habitat soil fungal sequencing DNA was extracted from the samples by the CTAB method, and the extracted DNA was detected by 1% agarose gel electrophoresis. PCR extraction was performed using barcode primers and high-fidelity enzymes. The following primer sequences were used: 5'-AAGCTCGTAGTTGAATTTCG-3' and 5'-CCCAACTATCCCTATTAATCAT-3' [35, 36]. The PCR products were mixed and detected by 2% agarose gel electrophoresis. The quantified PCR products were subjected to Illumina HiSeq sequencing. First, Trimmomatic (version 0.33) [37] was used to filter the quality of the original data, and then Cutadapt (version 1.9.1)[38] was used to identify and remove the primer sequences. Subsequently, USEARCH (version 10) was used to splice the double-end reads and remove the chimeras (UCHIME, version 8.1)[39]. Chimeric sequences were removed to finally obtain high-quality sequences, and the characteristic sequences were taxonomically annotated using the simple Bayes classifier with UNITE as the reference database[40]. Data analysis Soil nutrient difference analysis The soil nutrients of the three Paphiopedilum species were statistically analyzed by SPSS 22.0. Univariate analysis of variance was used to analyze the means. When the variance analysis results of soil nutrients between different species were significant at the p < 0.05 level, the least significant difference (LSD) test was used to compare the mean values of the soil variables. Analysis of fungal community structure in the habitat FLASH (v.1.2.7) was used to merge forward and reverse double-ended sequences from the MiSeq platform. A paired-end reading was obtained for each sample based on a unique barcode sequence. QIIME software was used to remove data clutter, and 97% similarity was used as the standard to divide operational taxonomic units. Usearch was used to remove chimeras, and the RDP classifier Bayesian algorithm was used to perform taxonomic analysis on representative OTU sequences. Species classification was performed using the fungal database Unite8.0/Fungi (Unite Release 8.0) for comparison and identification. In this study, the abundance of fungal species was analyzed based on the Euclidean distance algorithm, and the FUNGuild online database platform was used to predict the function of soil fungi in three Paphiopedilum species. The Chao1 index, ACE index, Shannon index and Simpson index were used to represent the alpha diversity index, and a t test was used to determine significant differences. ANOSIM was used to verify the reliability of the species grouping. Beta diversity was analyzed by nonmetric multidimensional scaling (NMDS) calculated by the Bray‒Curtis distance, and the results were subjected to the Adonis nonparametric variance test. The above analysis was implemented using the BioCloud platform (https://www.biocloud.net/). To effectively reveal the symbiotic network relationships of the soil fungi in the three Paphiopedilum habitats , the soil fungal species data of the Paphiopedilum plant habitats were filtered under conditions of a relative abundance ≥ 0.1% and at least three sampling points. Spearman correlation was performed on the filtered data set, and a correlation coefficient (ρ) ≥ 0.5 and p < 0.01 were selected to construct the network. The main topological attributes of the network were calculated by using the “igraph” package of R software, and the nodes were calculated and visualized with Gephi software. The Mantel test was used to explore the effects of habitat soil nutrients on fungal diversity. db-RDA was used to reveal the effects of habitat soil nutrients on soil fungal community changes. Spearman correlation analysis was used to reveal the correlation between habitat soil nutrients and dominant habitat soil fungal groups. The analysis and mapping were achieved using the “vegan” package, “ggcor” package, and “corrplot” package of R software. Results 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. Discussion Ex situ conservation, as an important measure for reducing the risk of orchid extinction, has always played a key role in biodiversity conservation [41,42]. However, the success of ex situ conservation depends on the understanding of the ecological habits of species and the mastery of the ecological and biological characteristics of the living environment of species. In particular, for species such as orchids that are highly dependent on fungal symbiosis, revealing the characteristics of their habitat soil environment is a necessary prerequisite for successful ex situ conservation. Although some studies have been carried out on the habitat of wild orchids, most of these studies have been based on the distribution pattern, habitat preference and habitat evaluation of orchids, ignoring the specific habitat characteristics of orchids [43-46]. In this study, targeted sampling methods were used to study the habitat of wild populations of Paphiopedilum species. The habitat characteristics, soil nutrients and soil fungal microbial community structures of three rare and endangered Paphiopedilum species were revealed, and the relationships among them were explored. This information will be helpful for the construction of the simulated environment and site selection for field return in future ex situ conservation processes. The results showed that the three species of Paphiopedilum preferred to grow in low-lying areas with high vegetation canopy density and ventilation, and the relative humidity of the habitat was relatively high, which made the Paphiopedilum species more resistant to arid climatic conditions; however, this negative terrain may also make it difficult for the population to spread. The soil physical and chemical properties of the three species of Paphiopedilum were not significantly different except for the significant differences in available nitrogen and available phosphorus, which may be related to the strong topographic heterogeneity in this area. A survey revealed that the three species of Paphiopedilum are distributed in a narrow area in the mountainous areas of Southwest China. Southwest China has both karst and nonkarst landforms, diverse soil-forming bedrock, an interwoven distribution of soil types, and a changeable climate and space-time heterogeneity. [47] The complexity of this environmental background has caused variations in soil nutrients in the habitats of Paphiopedilum species, which may lead to large differences in soil nutrients at different distribution points within the same species [48-50]. The soil fungal community is not only the main factor that directly affects plant growth and distribution but also the key bridge that indirectly affects plant growth and distribution. Its composition and structure are often controlled by environmental conditions[51,52]. In this study, the number of soil-specific OTUs in the P. emersonii habitat was greater than that in the other two species of Paphiopedilum , indicating that the soil-specific fungi in the P. emersonii habitat were more diverse and that the habitat characteristics of this growth area were related. [31] A survey revealed that the habitat of P. emersonii was mainly humus soil, which itself contains abundant fungi. However, P . wenshanense and P. armeniacum are distributed in high-elevation areas of Yunnan (1500-2000 m above sea level), which has a subtropical mountain monsoon climate characterized by long sunshine and low relative humidity, which limits the diversity of soil fungi. The soil composition and structure of the fungal communities of the three Paphiopedilum species differed at the phylum and genus levels, and their dominant fungal groups appeared. The differences in composition may be due to two reasons: on the one hand, the mycorrhizal characteristics of different Paphiopedilum species can screen out fungal groups that are symbiotic with Paphiopedilum species and change the fungal community structure of the habitat soil through alternate, antagonistic and competitive methods during the growth process [53-55]. On the other hand, there are differences in the growth environments of different Paphiopedilum species. In particular, the environmental heterogeneity caused by geographical distribution is a direct environmental factor affecting the composition of fungal communities in habitats[56,57]. To adapt to the environment, fungi adopt different nutritional methods, which is a survival strategy adopted by fungi to adapt to different living conditions [58,59]. In this study, saprophytic fungi and ectomycorrhizal fungi were dominant in the soil of the three Paphiopedilum species, providing a good material basis and symbiotic fungal resources for plant survival. It is generally believed that the diversity of fungal functional groups in the soil is related to the complexity of the environment [60,61], which may be related to the special habitat preferences of Paphiopedilum species. This group of plants loves to environments with high vegetation canopy density, good ventilation and appropriate shading and often grows in rock joints or humus layers near the bases of other woody plants. This habitat itself breeds abundant saprophytic fungi and ectomycorrhizal fungi. It plays an ecological role in plant growth promotion and nutrient cycling [62-65].Ectomycorrhizal groups may provide the initial impetus for the germination of Paphiopedilum seeds and are also the main source of heterotrophic fungi in the early stage of Paphiopedilum species. Saprophytic fungi accelerate the circulation of habitat materials, soften the soil texture, and increase water permeability and air permeability [66,67] so that nutritional conditions and habitat characteristics are more conducive to the growth of Paphiopedilum species. We also found a small amount of orchid mycorrhizal fungi in the habitat soil. [68] indicated that when the habitat soil fungal community provides the original material for orchid mycorrhizal fungi, mycorrhizal fungi will also have a place in the habitat soil with the growth of Paphiopedilum , and this result will also be conducive to the germination of orchid seeds. [69,70]. The alpha diversity index is an important measure of community characteristics in ecology and biological sciences. This study revealed that the habitat soil fungal diversity indices of three Paphiopedilum species were consistent with the results of detecting the number of fungal-specific OTUs, reflecting the consistency between fungal community diversity and the number of fungal-specific OTUs. Beta diversity emphasizes the change in community structure along a gradient or the direction of variation within a specific gradient range [71,72]. This study also confirmed that there are obvious differences in the habitat soil fungal community structure of the three Paphiopedilum species. Although the three Paphiopedilum species have similar genetic relationships [15], this may be due to differences in their historical geographical distributions and habitat conditions, which leads to changes in the soil fungal community structure in their habitats and reflects differences in habitat selection among the three Paphiopedilum species. A microbial co-occurrence network is a powerful means to reveal the coexistence relationship between microorganisms. This study revealed that the soil fungal habitat of the three Paphiopedilum species was dominated by positive effect symbiosis, and the proportion of negative effect symbiosis was very small, which is completely different from the results of biological co-occurrence network analysis of other research objects [73,74]. We speculate that the construction of soil fungal communities in these habitats of the three Paphiopedilum species has reached a mature level, and many fungi can coexist harmoniously and play a biological role together [75-77], which is also reflected in the diversity of habitat soil fungal functional groups of the three Paphiopedilum species. Of course, we speculate that this positive effect-based microbial coexistence model enables soil microorganisms to better resist the stress of adverse environments to provide nutrition for Paphiopedilum species in extreme environments[78]. This speculation needs to be further verified. The fungal community, which plays an important role in the soil environment, is also affected by soil nutrients [79-82]. This study revealed the effects of habitat-related nutrient changes on the soil fungal community structure of three Paphiopedilum species. The Mantel test revealed that the pH had a significant controlling effect on the diversity of the soil fungal communities of the three Paphiopedilum species. Soil pH controls diversity by directly affecting the survival, competition, growth and reproductive efficiency of soil fungi [83]. Moreover, the soil pH is also an indirect manifestation of differences in comprehensive environmental conditions [84]. Liu et al . confirmed that the soil pH is an important predictor of soil fungal groups in Southwest China [85]. In future ex situ conservation of Paphiopedilum species, attention should be given to monitoring the soil pH. This study also revealed the relationships between these dominant fungal groups and soil nutrients, and the compositions of the dominant habitat soil fungal groups of the three Paphiopedilum species were complex. There are Rozellomycota, Olpidiomycota, Mortierellomycota, Kickxellomycota, Glomeromycota, Entorrhizomycota, Chytridiomycota, Calcarisporielomycota, Basidiomycota, Ascomycota and other fungal groups. Most of the groups were significantly associated with soil nutrients, which is similar to previous research results[86,87]. That is, fluctuations in soil properties will cause changes in fungal community structure. Moreover, as an important microorganism in soil, fungi play a vital role in the material and energy cycle of soil systems and the improvement of soil structure through the decomposition of organic matter and the release of nutrients. This shows that soil nutrients are also a factor worth considering in future ex situ conservation. Conclusion The results of this study showed that the three species of Paphiopedilum grew in low-lying, shaded and ventilated places, and their habitat characteristics were highly heterogeneous. There was no significant difference in soil nutrients among the different species, but the soil nutrients at different distribution points of the same species had strong variability. The analysis of the soil fungal community structure among the habitats revealed that the soil fungal community composition significantly differed among the three Paphiopedilum species, but in the P. emersonii habitat soil, more unique OTUs and fungal species were detected. Similarly, the soil fungal functional groups of the three Paphiopedilum species were similar, and they were mainly composed of saprophytic, symbiotic and pathotrophic symbiotic fungi. These included UndefinedSaprotroph, Ectomycorrhizal, Undefined-Biotroph, Soil Saprotroph, Fungal Parasite, Wood Saprotroph, and Plant Saprophytic fungi. There were significant differences in the soil fungal communities among the three Paphiopedilum species. The Simpson index and Shannon index of P. emersonii were significantly greater than those of the other two species. The results of microbial co-occurrence network analysis showed that the symbiosis of soil fungi in the three Paphiopedilum species was mainly positive, and P. emersonii had a greater degree of symbiosis network modularity. Nutrients significantly affected the Shannon and Simpson indices of the three Paphiopedilum species. Soil nutrients represented a total of 80.70% of the horizontal changes in the dominant soil fungal groups of the three Paphiopedilum species. The main groups of soil fungi in each habitat of Paphiopedilum species were significantly correlated with soil nutrients, indicating that soil nutrients and soil fungal communities interacted with each other. Declarations Funding The project was commissioned by National Natural Foundation , China. Project Number: 32360101 Availability of data and materials Raw amplicon sequence data related to this study were deposited in the NCBI Sequence Read Archive (NCBI SRA) under Bioproject PRJNA888962. Given that the study’s subject ( P. armeniacum , P. emersonii , P.wenshanense ) belongs to rare or endangered species in the IUCN standard. We collected their habitat soil with the permission of the local conservation authority, and there was no damage to the plants. Ethics approval and consent to participate We complied with all relevant institutional, national and international guidelines in experimental research and field studies on plants. Material sampling done with permission by the Department of Wildlife Conservation and Nature Reserve Management of the National Forestry and Grassland Administration of China.The identification of the three species was completed by Professor An Mingtai. The habitat soil voucher specimens were collected in the Biodiversity Conservation and Research Center of Guizhou University, and the voucher specimens numbers were XH-2022,WS-2022, BH-2022. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Authors' contributions Li Tian wrote the main manuscript text and Feng Liu and Yang Zhang prepared figures 1-3. Mingtai An reviewed the manuscript. References Herrera H, García RI, Meneses C, Pereira G, Arriagada C: Orchid Mycorrhizal Interactions on the Pacific Side of the Andes from Chile. A Review . J SOIL SCI PLANT NUT 2019, 19 (1):187-202. Chengru L, Na D, Yamei Z, Shasha W, Zhongjian L, Junwen Z: A review for the breeding of orchids: Current achievements and prospects . HORTIC PLANT J 2021, 7 (05):380-392. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Berg C, Schuiteman A: updated classification of Orchidaceae . BOT J LINN SOC 2015, 177 (2):151-174. 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Wang Y, Wu F, Li X, Li C, Zhao Y, Gao Y, Liu J: Effects of plants and soil microorganisms on organic carbon and the relationship between carbon and nitrogen in constructed wetlands . ENVIRON SCI POLLUT R 2023, 30 (22):62249-62261. Additional Declarations No competing interests reported. Supplementary Files gelimages.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4245399","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294045889,"identity":"a36efa88-26aa-4ab0-9b70-318e19a15b43","order_by":0,"name":"Li Tian","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Tian","suffix":""},{"id":294045891,"identity":"2052cec4-8e8a-4b7f-8a61-57e748583a35","order_by":1,"name":"Mingtai An","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFCCBCA2ADEYGx8kVNiQoIWHgbHZ4MGZNGK1MIC0MLBJPmw7RFiDvHvys0c3Cu7Y7Wc/3FaRwHaAgb+9OwGvFsMzz8yNcwyeJffwJLbdSOC5wyBx5uwG/FpmJJhJ5xgcTuZhAGmReMZgIJFLSEv6N4gW/odtBQkGhwlrkZfIAdtixyOR2MaQkECEFgOeN2UgLQk8Nx42SyQcSOMh6Bf59vRt0jl/Dtuz96c//Pjzn40cf3svAVsOQOjEBqgAD17lYFugSu0JqhwFo2AUjIKRCwBQPUws/dbLJwAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Mingtai","middleName":"","lastName":"An","suffix":""},{"id":294045893,"identity":"4e7cba90-0262-4168-961a-4b1db59fbca3","order_by":2,"name":"Feng Liu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Liu","suffix":""},{"id":294045895,"identity":"17043c58-75f8-4000-9e94-fa21c0eb7063","order_by":3,"name":"Yang Zhang","email":"","orcid":"","institution":"Guiyang City, Guizhou Province Forestry Bureau","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-04-10 06:12:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4245399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4245399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55245361,"identity":"0d9b664e-3c2c-4316-843f-7a81516da3e7","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1371303,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing results of soil fungi in three Paphiopedilum habitats. (A) Dilution curves of soil fungi in three \u003cem\u003ePaphiopedilum \u003c/em\u003ehabitats. (B) Venn diagram showing the number of unique and common OTUs of the three species.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/a5c3c3d65f6da79f8358e197.jpg"},{"id":55245363,"identity":"ffc3e56e-97f9-4938-840f-7d6c03779357","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3419422,"visible":true,"origin":"","legend":"\u003cp\u003eThe main species composition of the soil fungal communities of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/9e966801021ceadda34ed942.jpg"},{"id":55245364,"identity":"ea2d67ac-d6cf-4088-95bb-4255a89f5e6f","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1309551,"visible":true,"origin":"","legend":"\u003cp\u003eComposition ratios of the soil fungal functional groups of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. (A) Commented on a nutritional basis. (B) Annotation by way of absorbing environmental resources.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/1e8113ea65f0ad82600506c0.jpg"},{"id":55245369,"identity":"8267a5f0-af95-4902-93b4-e8a7cf95754c","added_by":"auto","created_at":"2024-04-24 16:02:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1909179,"visible":true,"origin":"","legend":"\u003cp\u003eThe alpha diversity of fungi in the habitats of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. *and* * indicate that there were significant differences between diversity indices (p \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/80cf042de83e10bd698e3589.jpg"},{"id":55245818,"identity":"fe3bfd3a-9df0-4e5f-bf71-d3d15d3ba107","added_by":"auto","created_at":"2024-04-24 16:10:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":946310,"visible":true,"origin":"","legend":"\u003cp\u003eMANOVA analysis at the (a) gate level and (b) genus level\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/17e28ad678c5b30787f13133.jpg"},{"id":55245365,"identity":"58e47b97-0573-46ce-906e-157d996eb397","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1621299,"visible":true,"origin":"","legend":"\u003cp\u003eNonmetric multidimensional scaling analysis (NMDS) at the (a) gate level and (b) genus level. Each point in the figure represents a sample; different colors represent different groups; the elliptical ring represents a 95% confidence ellipse. When the stress is less than 0.1, it can be considered a good sort; when the stress is less than 0.05, it has good representativeness. It is generally believed that when the stress is less than 0.2, NMDS analysis has a certain reliability. The closer the sample is to the coordinate diagram, the greater the similarity.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/68fa6cbddc3f47afef073f50.jpg"},{"id":55245368,"identity":"1e522e8d-07de-4503-906d-a82ac3734ef7","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4445889,"visible":true,"origin":"","legend":"\u003cp\u003eSoil fungal co-occurrence network of three \u003cem\u003ePaphiopedilum \u003c/em\u003especies in habitats. Each node represents an OTU, and the connection of the nodes represents a strong correlation (Spearman'sr \u0026gt; 0.6), p \u0026lt; 0.01 adjusted by fdr. The edges between the nodes represent a significant correlation between the two OTUs. The red edge indicates a positive correlation, and the green edge indicates a negative correlation.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/cc01c5a4fa95349b852ee80f.jpg"},{"id":55245367,"identity":"6014f41f-5e5c-4d2a-9348-4cad04172a02","added_by":"auto","created_at":"2024-04-24 16:02:51","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2526447,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Mental test of alpha diversity and soil nutrients. (B) Redundancy analysis of the main soil fungal groups and soil nutrients.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/ef8a8a62758e9a460c2374eb.jpg"},{"id":57187286,"identity":"b60bdc0a-e818-46a4-8786-d25b206e6c31","added_by":"auto","created_at":"2024-05-27 06:19:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7383888,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/a7d4f067-f8e5-4591-a792-8e2ea954c0e9.pdf"},{"id":55245372,"identity":"686bb175-2e39-48fb-ae91-be50d949f8bd","added_by":"auto","created_at":"2024-04-24 16:02:52","extension":"zip","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":2856578,"visible":true,"origin":"","legend":"","description":"","filename":"gelimages.zip","url":"https://assets-eu.researchsquare.com/files/rs-4245399/v1/eb27631c0ee95c062ed3a13d.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China","fulltext":[{"header":"Background","content":"\u003cp\u003eOrchidaceae is one of the largest families of higher plants and one of the most evolutionarily morphologically rich plant families in nature. There are approximately 25,000-35,000 Orchidaceae species in 800 genera worldwide[1, 2]. Orchid organs are highly specialized, diverse, and highly adaptable to the environment and are widely distributed in various terrestrial ecosystems except for the poles and extreme arid desert regions[3, 4]. Orchids have highly specialized mycorrhizal communities and special pollination mechanisms [5] and are extremely sensitive to habitat changes [6, 7]. Orchids also have unique ornamental functions and medicinal value [8], making them \u0026ldquo;flagship\u0026rdquo; species in the ecological environment[9]. At present, due to severe anthropogenic activity and the impact of global climate change, wild orchids are facing great pressure to survive. All wild orchids in the world are protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and their trading and trading are explicitly prohibited [10]. \u003cem\u003ePaphiopedilum\u003c/em\u003e belongs to the Orchidaceae family and has large, slip-shaped lips on its petals [11, 12]. As the flower shape is similar to that of the slippers of noble European women in the Middle Ages, \u003cem\u003ePaphiopedilum\u003c/em\u003e is also known as the slipper orchid, which is the most ornamental group of orchaceae plants and enjoys a high reputation among international flower ornamentals. This topic has attracted the attention of many scientific researchers and flower lovers around the world [13]. Due to the ornamental value of \u003cem\u003ePaphiopedilum\u003c/em\u003e, the wild population is subject to predatory mining and the smuggling trade [14]. In addition, it is difficult for this group of plants to reproduce in the wild, and the species and number of plants are rapidly reduced or even extinct [15]. At present, all \u003cem\u003ePaphiopedilum\u003c/em\u003e species are listed on the CITES List and the IUCN Red List of endangered species, and in China, all of them are listed on the State Key Wild Plant Protection List, which is rare among the rare and endangered plant groups in the world [16]. Southwest China is the origin and differentiation center of \u003cem\u003ePaphiopedilum\u003c/em\u003e and the distribution hotspot of \u003cem\u003ePaphiopedilum\u003c/em\u003e. However, according to a recent survey of wild \u003cem\u003ePaphiopedilum\u003c/em\u003e plants, the plants of \u003cem\u003ePaphiopedilum\u003c/em\u003e in Southwest China are suffering devastating damage, especially \u003cem\u003ePaphiopedilum armeniacum\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eP. armeniacum\u003c/em\u003e), \u003cem\u003ePaphiopedilum wenshanense\u003c/em\u003e (\u003cem\u003eP. wenshanense\u003c/em\u003e) and \u003cem\u003ePaphiopedilum\u003c/em\u003e \u003cem\u003eemersonii\u003c/em\u003e (\u003cem\u003eP. emersonii\u003c/em\u003e), which are the most endangered[17]. At present, fewer than 10 natural distribution sites of these three \u003cem\u003ePaphiopedilum\u003c/em\u003e species have been found, and the latter habitats are at risk of degradation and loss. These three species of \u003cem\u003ePaphiopedilum\u003c/em\u003e are listed as small populations on the wild plant protection list in China, which means that there is a risk of extinction in the wild at any time, which also indicates the urgency and priority of protection.\u003c/p\u003e\n\u003cp\u003eUnderstanding the ecological habits of orchids is a prerequisite for scientific conservation work, especially for ex situ conservation of endangered species [18, 19]. In the natural environment, because the seeds of orchids are small, the germ is degraded and lacks nutrients, and the germination of seeds depends on the help of fungi, which can provide necessary resources such as carbon, nitrogen and phosphorus [20]. The entire growth cycle of orchids also depends on compatible mycorrhizal fungi in the soil, whether specialized or compatible. Considering the heavy dependence of orchids on mycorrhizal fungi, it can be predicted that their spatial distribution largely depends on the spatial distribution of microorganisms in the soil of the habitat, and the composition of fungi in the soil of the habitat is a key feature of the ecological habits of orchids [21, 22]. There is evidence that changes in mycorrhizal fungal communities driven by habitat conditions can directly or indirectly affect the distribution dynamics of terrestrial orchids [23, 24]. In addition, some seed introduction experiments showed that the seeds could germinate outside the densely distributed areas of orchids, indicating that orchid seeds may recruit fungi from the soil of the habitat to complete symbiosis, germination and growth [25, 26] and gradually form a microbial community with orchid roots as the distribution core. Therefore, the soil fungal community in a habitat may be the origin of orchid mycorrhizal fungi [27-29]. Moreover, locations close to orchid plant growth represent fungal hotspots for seed germination and new plant establishment. However, most of the current studies have focused on the endophytic fungi and rhizosphere fungal communities of orchids [30-33] while ignoring the fungal communities in habitat soil. This lack of understanding of the species source and process of orchid mycorrhizal fungal community construction restricts the development of simulated environment construction for ex situ conservation. At present, due to the reduction in the number of remaining wild populations and the degradation of habitats, the supplementation of seedlings is difficult to guarantee, and the survival of wild populations is at stake. In this regard, a necessary and urgent solution is to construct a suitable habitat for the growth of \u003cem\u003ePaphiopedilum\u003c/em\u003e in a good artificial environment, expand its population size, and ultimately return to the wild. To ensure success, it is crucial to fully reveal the soil fungal communities in the last remaining habitat of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species, including how they change spatially and respond to the biotic environment.\u003c/p\u003e\n\u003cp\u003eIn this study, \u003cem\u003eP. armeniacum\u003c/em\u003e,\u003cem\u003eP. wenshanense\u003c/em\u003e,\u003cem\u003eP. emersonii\u003c/em\u003e, were used as research objects to comprehensively investigate the environmental characteristics of the remaining habitats, and soil samples were collected from the habitats of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e populations. The soil fungi in their habitats were determined based on high-throughput sequencing methods to clarify the fungal community composition structure, fungal functional groups, and fungal community diversity. The dynamic changes in fungi in the soil of \u003cem\u003ePaphiopedilum\u003c/em\u003e habitat were revealed, and the response of the fungal community structure in the habitat to the biotic environment was initially explored, compensating for the lack of research on the habitat characteristics of the wild population of this group of species. In addition. This study can also provide a reference for ex situ conservation, artificial breeding environment selection and the construction of this group of plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003cstrong\u003e collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, after fully investigating the natural distribution of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species, three natural distribution points with good growth and no man-made damage were selected for each species (Fig. 1). \u003cem\u003eP. armeniacum\u003c/em\u003e is distributed in Nujiang Prefecture, Yunnan Province. \u003cem\u003eP. wenshanense\u003c/em\u003e is distributed in Wenshan Prefecture, Yunnan Province. \u003cem\u003eP. emersonii\u003c/em\u003e is located at the junction of Guizhou Province and Guangxi Province. First, the last habitat of \u003cem\u003ePaphiopedilum\u003c/em\u003e was observed, and information on the latitude, longitude, altitude, slope, aspect and vegetation type was recorded. Sampling was carried out at 10 cm outside the dense distribution area of \u003cem\u003ePaphiopedilum\u003c/em\u003e species. We set the direction of possible landing of seeds as the sampling direction and extracted 20 grams of repeated soil samples in 3 directions at 5 cm from the soil layer. Soil fungal samples from 27 habitats of three species were collected. Before sampling, the air environment was disinfected with 75% medical alcohol to remove the undecomposed litter. To collect representative soil samples, the soil was transferred to a 4 \u0026deg;C refrigerator within 12 hours after sampling to minimize DNA degradation. For meta-barcode analysis, a small amount of soil (\u0026lt; 250 mg) was added to BashingBead TM Lysis Tubes (Zimo Research, Cambridge Bioscience, Cambridge, UK) to preserve environmental DNA for the extraction and amplification process. Soil nutrient test samples According to the above sampling method, 200 g of soil (at a depth of 0 ~ 10 cm) from each habitat was collected, placed in a sampling bag and returned to the laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil nutrient analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe soil physiochemical properties were analyzed according to Bao and are described briefly as follows[34]. The pH was determined by the water:soil = 2.5:1 extraction pH meter (PHS-3G) method. The water content was determined by the 105 \u0026deg;C drying\u0026ndash;weighing method. The organic carbon content was determined by the KCr2O7-H2SO4 external heating method. Total nitrogen was determined by the semimicro Kjeldahl method; total phosphorus was determined by the HClO4-H2SO4 digestion-molybdenum antimony colorimetric method. Total potassium was determined by NaOH melting-flame spectrophotometry. Available phosphorus was determined by a 0.03 mol/L NH4F-0.025 mol/L HCl extraction-molybdenum antimony anti-colorimetric method. Available potassium was extracted by a 1 mol/L neutral NH4OAC-flame photometer. Ammonium nitrogen and nitrate nitrogen were determined by a 2 mol/L KCl extraction-continuous flow analyzer. The soil enzyme activities were determined using an enzyme analysis kit (Yangling Xinhua Ecological Technology Co., Ltd., Shanxi, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHabitat soil fungal sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from the samples by the CTAB method, and the extracted DNA was detected by 1% agarose gel electrophoresis. PCR extraction was performed using barcode primers and high-fidelity enzymes. The following primer sequences were used: 5\u0026apos;-AAGCTCGTAGTTGAATTTCG-3\u0026apos; and 5\u0026apos;-CCCAACTATCCCTATTAATCAT-3\u0026apos; [35, 36]. The PCR products were mixed and detected by 2% agarose gel electrophoresis. The quantified PCR products were subjected to Illumina HiSeq sequencing. First, Trimmomatic (version 0.33) [37] was used to filter the quality of the original data, and then Cutadapt (version 1.9.1)[38] was used to identify and remove the primer sequences. Subsequently, USEARCH (version 10) was used to splice the double-end reads and remove the chimeras (UCHIME, version 8.1)[39]. Chimeric sequences were removed to finally obtain high-quality sequences, and the characteristic sequences were taxonomically annotated using the simple Bayes classifier with UNITE as the reference database[40].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData \u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil nutrient difference analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe soil nutrients of the three\u003cem\u003e Paphiopedilum\u003c/em\u003e species were statistically analyzed by SPSS 22.0. Univariate analysis of variance was used to analyze the means. When the variance analysis results of soil nutrients between different species were significant at the p \u0026lt; 0.05 level, the least significant difference (LSD) test was used to compare the mean values of the soil variables.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of fungal community structure in the habitat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFLASH (v.1.2.7) was used to merge forward and reverse double-ended sequences from the MiSeq platform. A paired-end reading was obtained for each sample based on a unique barcode sequence. QIIME software was used to remove data clutter, and 97% similarity was used as the standard to divide operational taxonomic units. Usearch was used to remove chimeras, and the RDP classifier Bayesian algorithm was used to perform taxonomic analysis on representative OTU sequences. Species classification was performed using the fungal database Unite8.0/Fungi (Unite Release 8.0) for comparison and identification.\u003c/p\u003e\n\u003cp\u003eIn this study, the abundance of fungal species was analyzed based on the Euclidean distance algorithm, and the FUNGuild online database platform was used to predict the function of soil fungi in three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. The Chao1 index, ACE index, Shannon index and Simpson index were used to represent the alpha diversity index, and a t test was used to determine significant differences. ANOSIM was used to verify the reliability of the species grouping. Beta diversity was analyzed by nonmetric multidimensional scaling (NMDS) calculated by the Bray‒Curtis distance, and the results were subjected to the Adonis nonparametric variance test. The above analysis was implemented using the BioCloud platform (https://www.biocloud.net/).\u003c/p\u003e\n\u003cp\u003eTo effectively reveal the symbiotic network relationships of the soil fungi in the three \u003cem\u003ePaphiopedilum habitats\u003c/em\u003e, the soil fungal species data of the Paphiopedilum plant habitats were filtered under conditions of a relative abundance \u0026ge; 0.1% and at least three sampling points. Spearman correlation was performed on the filtered data set, and a correlation coefficient (\u0026rho;) \u0026ge; 0.5 and p \u0026lt; 0.01 were selected to construct the network. The main topological attributes of the network were calculated by using the \u0026ldquo;igraph\u0026rdquo; package of R software, and the nodes were calculated and visualized with Gephi software.\u003c/p\u003e\n\u003cp\u003eThe Mantel test was used to explore the effects of habitat soil nutrients on fungal diversity. db-RDA was used to reveal the effects of habitat soil nutrients on soil fungal community changes. Spearman correlation analysis was used to reveal the correlation between habitat soil nutrients and dominant habitat soil fungal groups. The analysis and mapping were achieved using the \u0026ldquo;vegan\u0026rdquo; package, \u0026ldquo;ggcor\u0026rdquo; package, and \u0026ldquo;corrplot\u0026rdquo; package of R software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHabitat information and soil nutrient characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA habitat survey of three wild \u003cem\u003ePaphiopedilum\u003c/em\u003e species revealed that (Table 1) \u003cem\u003eP. armeniacum\u003c/em\u003e grows in mountain shrubs at an altitude of approximately 1700-2000 m, and the slope is usually 30-55\u0026deg;. \u003cem\u003eP. emersonii\u003c/em\u003e 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\u0026deg;). \u003cem\u003eP. wenshanense\u003c/em\u003e 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\u003cem\u003e\u0026nbsp;Paphiopedilum\u003c/em\u003e 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 \u003cem\u003eP. emersonii\u003c/em\u003e was significantly greater than that in the soil of \u003cem\u003eP. wenshanense\u003c/em\u003e. The available phosphorus in the soil of \u003cem\u003eP. emersonii\u003c/em\u003e and \u003cem\u003eP. armeniacum\u003c/em\u003e was significantly greater than that in the soil of \u003cem\u003eP. wenshanense\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTable 1 Habitat information of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"588\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.250425894378193%\"\u003e\n \u003cp\u003eSpecie\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.221465076660989%\"\u003e\n \u003cp\u003eAltitude（m）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.05792163543441%\"\u003e\n \u003cp\u003eGeomorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.458262350936968%\"\u003e\n \u003cp\u003eVegetational Form\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.90289608177172%\"\u003e\n \u003cp\u003eSlope（\u0026deg;）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.287904599659285%\"\u003e\n \u003cp\u003eAspect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.821124361158432%\"\u003e\n \u003cp\u003eMicrohabitat\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.250425894378193%\"\u003e\n \u003cp\u003e\u003cem\u003eP. armeniacum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.221465076660989%\"\u003e\n \u003cp\u003e1776-2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.05792163543441%\"\u003e\n \u003cp\u003eNormal Geomorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.458262350936968%\"\u003e\n \u003cp\u003eShrubwood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.90289608177172%\"\u003e\n \u003cp\u003e30-55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.287904599659285%\"\u003e\n \u003cp\u003eNorth‒West、North\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.821124361158432%\"\u003e\n \u003cp\u003eTree Root、Swallet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.250425894378193%\"\u003e\n \u003cp\u003e\u003cem\u003eP. emersonii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.221465076660989%\"\u003e\n \u003cp\u003e389-835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.05792163543441%\"\u003e\n \u003cp\u003eKarst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.458262350936968%\"\u003e\n \u003cp\u003eEvergreen And Deciduous Broad Leaved Forest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.90289608177172%\"\u003e\n \u003cp\u003e75-90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.287904599659285%\"\u003e\n \u003cp\u003eNorth South East West\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.821124361158432%\"\u003e\n \u003cp\u003eStonewall\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.250425894378193%\"\u003e\n \u003cp\u003e\u003cem\u003eP. wenshanense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.221465076660989%\"\u003e\n \u003cp\u003e1525-1630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.05792163543441%\"\u003e\n \u003cp\u003eKarst，Normal Geomorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.458262350936968%\"\u003e\n \u003cp\u003eShrubwood\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.90289608177172%\"\u003e\n \u003cp\u003e30-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.287904599659285%\"\u003e\n \u003cp\u003eNorthwest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.821124361158432%\"\u003e\n \u003cp\u003eTree Roots, Stone Crevices\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2 Differences in the soil nutrients in the habitats of the three \u003cem\u003epaphiopedilum\u003c/em\u003e species\u003c/p\u003e\n\u003cdiv style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"border: none;width:437.3pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:.2in;\"\u003e\n \u003cp style=\"margin:0in;text-align:left;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:.2in;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cem\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eP.armeniacum\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:.2in;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cem\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eP.wenshanense\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:.2in;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cem\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eP.emersonii\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTN\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e5.19\u0026plusmn;1.28a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e1.81\u0026plusmn;1.58a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e7.88\u0026plusmn;5.06a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;background:white;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e0.57\u0026plusmn;0.13a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;background:white;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eTK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e223.58\u0026plusmn;42.92ab\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e102.95\u0026plusmn;80.55b\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e316.74\u0026plusmn;85.20a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;background:white;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAP\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e4.1\u0026plusmn;2.19a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e1.08\u0026plusmn;0.08b\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e5.04\u0026plusmn;1.30a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;background:white;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003eAK\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e138.67\u0026plusmn;45.24a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e194.33\u0026plusmn;122.15a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e70\u0026plusmn;41.04a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:134.7pt;border:none;border-bottom:solid windowtext 1.0pt;background:white;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:black;'\u003epH\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e6.76\u0026plusmn;1.046a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:101.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e7.27\u0026plusmn;0.62a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.8pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;margin-left:-7.05pt;line-height:200%;\"\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;color:#993300;'\u003e7.77\u0026plusmn;0.32a\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: The values are the means \u0026plusmn; standard errors. Different lowercase letters in the same row indicate significant differences between horizontal gradients (P \u0026lt; 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\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies dilution curve and Venn diagram\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003eP. emersonii\u003c/em\u003e (Fig. 2b), followed by \u003cem\u003eP. wenshanense\u003c/em\u003e (n=232) and\u003cem\u003e\u0026nbsp;P.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003earmeniacum\u003c/em\u003e (n=211). There were 65 OTUs in the \u003cem\u003eP. wenshanense\u003c/em\u003e and \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003earmeniacum\u003c/em\u003e\u003cem\u003e\u0026nbsp;habitat soils\u003c/em\u003e, 25 OTUs in the \u003cem\u003eP.\u0026nbsp;\u003c/em\u003e\u003cem\u003earmeniacum\u003c/em\u003e and \u003cem\u003eP. emersonii\u003c/em\u003e habitat soils, and 46 OTUs in the \u003cem\u003eP. emersonii\u003c/em\u003e and \u003cem\u003eP. wenshanense\u003c/em\u003e habitat soils; moreover, there were 37 common OTUs in the three \u003cem\u003ePaphiopedilum\u003c/em\u003e habitat soils.\u003c/p\u003e\n\u003cp\u003eAccording 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 \u003cem\u003eP.\u003c/em\u003e \u003cem\u003eemersonii\u003c/em\u003e. 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 \u003cem\u003eP. armeniacum\u003c/em\u003e. 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 \u003cem\u003eP. wenshanense\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTable 3 Number of fungal species in the soil of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"563\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.404255319148938%\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003ePhylum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003eClass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003eOrder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003efamily\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003eGenus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003eOTUS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.404255319148938%\"\u003e\n \u003cp\u003e\u003cem\u003eP.emersonii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e127\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e560\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.404255319148938%\"\u003e\n \u003cp\u003e\u003cem\u003eP.armeniacum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e338\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.404255319148938%\"\u003e\n \u003cp\u003e\u003cem\u003eP.wenshanense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.404255319148938%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.815602836879433%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.99290780141844%\"\u003e\n \u003cp\u003e1068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFungi composition and functional group composition in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehabitat soil of three \u003cem\u003epaphiopedilum\u003c/em\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative abundance of fungal groups in the habitat soil of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species at the phylum and genus levels is shown in Fig. 3. The dominant group of fungi in the soil of \u003cem\u003eP. wenshanense\u003c/em\u003e was Calcarisporiellomycota, and no obvious dominant group was found in the soil of \u003cem\u003eP. armeniacum\u003c/em\u003e. There were dominant fungal groups, such as Kickxellomycota, Entorrhizomycota, Olpidiomycota and Rozellomycota, in the \u003cem\u003eP.\u0026nbsp;\u003c/em\u003e\u003cem\u003eemersonii\u003c/em\u003e\u003cem\u003e\u0026nbsp;habitat\u003c/em\u003e. At the genus level, there were unclassified_Sordariomycetes, \u003cem\u003eSebacina\u003c/em\u003e, unclassified_Basidiomycota, \u003cem\u003eBoletus\u003c/em\u003e, unclassified_Boletaceae, \u003cem\u003eArchaeorhizomyces\u003c/em\u003e and other dominant fungi in the habitat soil of \u003cem\u003eP. wenshanense\u003c/em\u003e. Unclassified _ Thelephoraceae, \u003cem\u003eHygrocybe\u003c/em\u003e, unclassified_Serendipitaceae, unclassified_Ascomycota, \u003cem\u003eTomentella\u003c/em\u003e and unclassified_Agaricomycetes were found in the soil fungal habitat of \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003earmeniacum\u003c/em\u003e. Acremonium, unclassified_Fungi, unclassified_Chaetothyriales, Apodus, unidentified, unclassified_Hypocreales and other soil fungi were found in the soil of \u003cem\u003eP. emersonii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eBased on the ecological role of fungi in the environment, the functional classification and annotation of soil fungi in the soil of three \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003eP. armeniacum\u003c/em\u003e accounted for 98%. It also accounts for more than 80% of the habitat of \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;wenshanense\u003c/em\u003e and \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;emersonii\u003c/em\u003e. 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;armeniacum\u003c/em\u003e, accounting for more than 95% of the relative abundance, and some orchid mycorrhizal and animal pathogenic fungi. The main fungal functional groups of \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e\u0026nbsp;wenshanense\u0026nbsp;\u003c/em\u003ein the soil habitat\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere 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 \u003cem\u003eP. emersonii\u0026nbsp;\u003c/em\u003ewere also rich, and their relative abundances were relatively similar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiversity\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe alpha diversity analysis of the habitat soil fungal communities of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e. The Simpson index and Shannon index of \u003cem\u003eP. emersonii\u003c/em\u003e were significantly greater than those of \u003cem\u003eP. armeniacum\u003c/em\u003e and significantly greater than those of \u003cem\u003eP. wenshanense\u003c/em\u003e(Fig 5). In addition, there was no significant difference in the four diversity indices between \u003cem\u003eP. armeniacum\u003c/em\u003e and \u003cem\u003eP. wenshanense\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo clarify the overall differences in the soil fungal community structure among the three \u003cem\u003ePaphiopedilum\u003c/em\u003e 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eFigure 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\u003cem\u003e\u0026nbsp;Paphiopedilum\u003c/em\u003e 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 \u003cem\u003ePaphiopedilum\u003c/em\u003e species were more obvious at the phylum level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;fungal co-occurrence network of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the potential interactions between the soil fungi in the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species and the changes in the co-occurrence network, an OTU-level co-occurrence network of the soil fungi of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species was constructed based on random matrix theory. The same threshold (r \u0026gt; 0.6, p \u0026lt; 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 \u003cem\u003eP. emersonii\u003c/em\u003e, 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 \u003cem\u003eP. armeniacum\u003c/em\u003e. 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 \u003cem\u003eP. wenshanense\u003c/em\u003e 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 \u003cem\u003eP. emersonii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. armeniacum\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelationships\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;between habitat soil fungi and soil nutrients of three \u003cem\u003epaphiopedilum\u003c/em\u003e species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u003cem\u003eP. emersonii\u003c/em\u003e\u003cem\u003e\u0026nbsp;habitat\u003c/em\u003e, 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 \u003cem\u003eP. armeniacum\u003c/em\u003e, 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 \u003cem\u003eP. wenshanense habitat\u003c/em\u003e, 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.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEx situ conservation, as an important measure for reducing the risk of orchid extinction, has always played a key role in biodiversity conservation [41,42]. However, the success of ex situ conservation depends on the understanding of the ecological habits of species and the mastery of the ecological and biological characteristics of the living environment of species. In particular, for species such as orchids that are highly dependent on fungal symbiosis, revealing the characteristics of their habitat soil environment is a necessary prerequisite for successful ex situ conservation. Although some studies have been carried out on the habitat of wild orchids, most of these studies have been based on the distribution pattern, habitat preference and habitat evaluation of orchids, ignoring the specific habitat characteristics of orchids [43-46]. In this study, targeted sampling methods were used to study the habitat of wild populations of \u003cem\u003ePaphiopedilum\u003c/em\u003e species. The habitat characteristics, soil nutrients and soil fungal microbial community structures of three rare and endangered \u003cem\u003ePaphiopedilum\u003c/em\u003e species were revealed, and the relationships among them were explored. This information will be helpful for the construction of the simulated environment and site selection for field return in future ex situ conservation processes.\u003c/p\u003e\n\u003cp\u003eThe results showed that the three species of \u003cem\u003ePaphiopedilum\u003c/em\u003e preferred to grow in low-lying areas with high vegetation canopy density and ventilation, and the relative humidity of the habitat was relatively high, which made the \u003cem\u003ePaphiopedilum\u003c/em\u003e species more resistant to arid climatic conditions; however, this negative terrain may also make it difficult for the population to spread. The soil physical and chemical properties of the three species of \u003cem\u003ePaphiopedilum\u003c/em\u003e were not significantly different except for the significant differences in available nitrogen and available phosphorus, which may be related to the strong topographic heterogeneity in this area. A survey revealed that the three species of \u003cem\u003ePaphiopedilum\u003c/em\u003e are distributed in a narrow area in the mountainous areas of Southwest China. Southwest China has both karst and nonkarst landforms, diverse soil-forming bedrock, an interwoven distribution of soil types, and a changeable climate and space-time heterogeneity. [47] The complexity of this environmental background has caused variations in soil nutrients in the habitats of \u003cem\u003ePaphiopedilum\u003c/em\u003e species, which may lead to large differences in soil nutrients at different distribution points within the same species [48-50]. The soil fungal community is not only the main factor that directly affects plant growth and distribution but also the key bridge that indirectly affects plant growth and distribution. Its composition and structure are often controlled by environmental conditions[51,52].\u003c/p\u003e\n\u003cp\u003eIn this study, the number of soil-specific OTUs in the \u003cem\u003eP. emersonii\u003c/em\u003e habitat was greater than that in the other two species of \u003cem\u003ePaphiopedilum\u003c/em\u003e, indicating that the soil-specific fungi in the \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003eemersonii\u003c/em\u003e habitat were more diverse and that the habitat characteristics of this growth area were related. [31] A survey revealed that the habitat of \u003cem\u003eP.\u003c/em\u003e emersonii was mainly humus soil, which itself contains abundant fungi. However, \u003cem\u003eP\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e wenshanense\u003c/em\u003e and \u003cem\u003eP.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003earmeniacum\u003c/em\u003e are distributed in high-elevation areas of Yunnan (1500-2000 m above sea level), which has a subtropical mountain monsoon climate characterized by long sunshine and low relative humidity, which limits the diversity of soil fungi. The soil composition and structure of the fungal communities of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species differed at the phylum and genus levels, and their dominant fungal groups appeared. The differences in composition may be due to two reasons: on the one hand, the mycorrhizal characteristics of different \u003cem\u003ePaphiopedilum\u003c/em\u003e species can screen out fungal groups that are symbiotic with \u003cem\u003ePaphiopedilum\u003c/em\u003e species and change the fungal community structure of the habitat soil through alternate, antagonistic and competitive methods during the growth process [53-55]. On the other hand, there are differences in the growth environments of different \u003cem\u003ePaphiopedilum\u003c/em\u003e species. In particular, the environmental heterogeneity caused by geographical distribution is a direct environmental factor affecting the composition of fungal communities in habitats[56,57]. To adapt to the environment, fungi adopt different nutritional methods, which is a survival strategy adopted by fungi to adapt to different living conditions [58,59]. In this study, saprophytic fungi and ectomycorrhizal fungi were dominant in the soil of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species, providing a good material basis and symbiotic fungal resources for plant survival. It is generally believed that the diversity of fungal functional groups in the soil is related to the complexity of the environment [60,61], which may be related to the special habitat preferences of \u003cem\u003ePaphiopedilum\u003c/em\u003e species. This group of plants loves to environments with high vegetation canopy density, good ventilation and appropriate shading and often grows in rock joints or humus layers near the bases of other woody plants. This habitat itself breeds abundant saprophytic fungi and ectomycorrhizal fungi. It plays an ecological role in plant growth promotion and nutrient cycling [62-65].Ectomycorrhizal groups may provide the initial impetus for the germination of \u003cem\u003ePaphiopedilum\u003c/em\u003e seeds and are also the main source of heterotrophic fungi in the early stage of \u003cem\u003ePaphiopedilum\u003c/em\u003e species. Saprophytic fungi accelerate the circulation of habitat materials, soften the soil texture, and increase water permeability and air permeability [66,67] so that nutritional conditions and habitat characteristics are more conducive to the growth of \u003cem\u003ePaphiopedilum\u003c/em\u003e species. We also found a small amount of orchid mycorrhizal fungi in the habitat soil. [68] indicated that when the habitat soil fungal community provides the original material for orchid mycorrhizal fungi, mycorrhizal fungi will also have a place in the habitat soil with the growth of \u003cem\u003ePaphiopedilum\u003c/em\u003e, and this result will also be conducive to the germination of orchid seeds. [69,70].\u003c/p\u003e\n\u003cp\u003eThe alpha diversity index is an important measure of community characteristics in ecology and biological sciences. This study revealed that the habitat soil fungal diversity indices of three \u003cem\u003ePaphiopedilum \u003c/em\u003especies were consistent with the results of detecting the number of fungal-specific OTUs, reflecting the consistency between fungal community diversity and the number of fungal-specific OTUs. Beta diversity emphasizes the change in community structure along a gradient or the direction of variation within a specific gradient range [71,72]. This study also confirmed that there are obvious differences in the habitat soil fungal community structure of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. Although the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species have similar genetic relationships [15], this may be due to differences in their historical geographical distributions and habitat conditions, which leads to changes in the soil fungal community structure in their habitats and reflects differences in habitat selection among the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. A microbial co-occurrence network is a powerful means to reveal the coexistence relationship between microorganisms. This study revealed that the soil fungal habitat of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species was dominated by positive effect symbiosis, and the proportion of negative effect symbiosis was very small, which is completely different from the results of biological co-occurrence network analysis of other research objects [73,74]. We speculate that the construction of soil fungal communities in these habitats of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species has reached a mature level, and many fungi can coexist harmoniously and play a biological role together [75-77], which is also reflected in the diversity of habitat soil fungal functional groups of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. Of course, we speculate that this positive effect-based microbial coexistence model enables soil microorganisms to better resist the stress of adverse environments to provide nutrition for \u003cem\u003ePaphiopedilum\u003c/em\u003e species in extreme environments[78]. This speculation needs to be further verified.\u003c/p\u003e\n\u003cp\u003eThe fungal community, which plays an important role in the soil environment, is also affected by soil nutrients [79-82]. This study revealed the effects of habitat-related nutrient changes on the soil fungal community structure of three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. The Mantel test revealed that the pH had a significant controlling effect on the diversity of the soil fungal communities of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. Soil pH controls diversity by directly affecting the survival, competition, growth and reproductive efficiency of soil fungi [83]. Moreover, the soil pH is also an indirect manifestation of differences in comprehensive environmental conditions [84]. Liu \u003cem\u003eet al\u003c/em\u003e. confirmed that the soil pH is an important predictor of soil fungal groups in Southwest China [85]. In future ex situ conservation of \u003cem\u003ePaphiopedilum\u003c/em\u003e species, attention should be given to monitoring the soil pH. This study also revealed the relationships between these dominant fungal groups and soil nutrients, and the compositions of the dominant habitat soil fungal groups of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species were complex. There are Rozellomycota, Olpidiomycota, Mortierellomycota, Kickxellomycota, Glomeromycota, Entorrhizomycota, Chytridiomycota, Calcarisporielomycota, Basidiomycota, Ascomycota and other fungal groups. Most of the groups were significantly associated with soil nutrients, which is similar to previous research results[86,87]. That is, fluctuations in soil properties will cause changes in fungal community structure. Moreover, as an important microorganism in soil, fungi play a vital role in the material and energy cycle of soil systems and the improvement of soil structure through the decomposition of organic matter and the release of nutrients. This shows that soil nutrients are also a factor worth considering in future ex situ conservation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this study showed that the three species of \u003cem\u003ePaphiopedilum\u003c/em\u003e grew in low-lying, shaded and ventilated places, and their habitat characteristics were highly heterogeneous. There was no significant difference in soil nutrients among the different species, but the soil nutrients at different distribution points of the same species had strong variability. The analysis of the soil fungal community structure among the habitats revealed that the soil fungal community composition significantly differed among the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species, but in the \u003cem\u003eP. emersonii\u003c/em\u003e habitat soil, more unique OTUs and fungal species were detected. Similarly, the soil fungal functional groups of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species were similar, and they were mainly composed of saprophytic, symbiotic and pathotrophic symbiotic fungi. These included UndefinedSaprotroph, Ectomycorrhizal, Undefined-Biotroph, Soil Saprotroph, Fungal Parasite, Wood Saprotroph, and Plant Saprophytic fungi. There were significant differences in the soil fungal communities among the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. The Simpson index and Shannon index of \u003cem\u003eP. emersonii\u003c/em\u003e were significantly greater than those of the other two species. The results of microbial co-occurrence network analysis showed that the symbiosis of soil fungi in the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species was mainly positive, and \u003cem\u003eP. emersonii\u003c/em\u003e had a greater degree of symbiosis network modularity. Nutrients significantly affected the Shannon and Simpson indices of the three\u003cem\u003e\u0026nbsp;Paphiopedilum\u003c/em\u003e species. Soil nutrients represented a total of 80.70% of the horizontal changes in the dominant soil fungal groups of the three \u003cem\u003ePaphiopedilum\u003c/em\u003e species. The main groups of soil fungi in each habitat of \u003cem\u003ePaphiopedilum\u003c/em\u003e species were significantly correlated with soil nutrients, indicating that soil nutrients and soil fungal communities interacted with each other.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was commissioned by National Natural Foundation , China. Project Number: 32360101\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw amplicon sequence data related to this study were deposited in the NCBI Sequence Read Archive (NCBI SRA) under Bioproject PRJNA888962. Given that the study\u0026rsquo;s subject (\u003cem\u003eP. armeniacum\u003c/em\u003e,\u003cem\u003eP. emersonii\u003c/em\u003e, \u003cem\u003eP.wenshanense\u003c/em\u003e) belongs to rare or endangered species in the IUCN standard. We collected their habitat soil with the permission of the local conservation authority, and there was no damage to the plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe complied with all relevant institutional, national and international guidelines in experimental research and field studies on plants. Material sampling done with permission by the Department of Wildlife Conservation and Nature Reserve Management of the National Forestry and Grassland Administration of China.The identification of the three species was completed by Professor An Mingtai. The habitat soil voucher specimens were collected in the Biodiversity Conservation and Research Center of Guizhou University, and the voucher specimens numbers were XH-2022,WS-2022, BH-2022.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Tian wrote the main manuscript text and Feng Liu and Yang Zhang prepared figures 1-3. Mingtai An reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHerrera H, Garc\u0026iacute;a RI, Meneses C, Pereira G, Arriagada C: \u003cstrong\u003eOrchid Mycorrhizal Interactions on the Pacific Side of the Andes from Chile. 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\u003cstrong\u003e30\u003c/strong\u003e(22):62249-62261.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Orchid, Paphiopedilum, Soil fungi, Habitat characteristics, Ex situ conservation","lastPublishedDoi":"10.21203/rs.3.rs-4245399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4245399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e \u003cem\u003ePaphiopedilum armeniacum\u003c/em\u003e, \u003cem\u003ePaphiopedilum wenshanense\u003c/em\u003e and \u003cem\u003ePaphiopedilum emersonii\u003c/em\u003e are critically endangered wild orchids. The population is threatened, and the number of natural distribution sites has plummeted. Ex situ conservation and artificial breeding are the keys to maintaining the population to ensure the success of ex situ conservation and field return in the future. The habitat characteristics and soil nutrient information of the last remaining wild distribution sites of the three species were studied. ITS high-throughput sequencing was used to reveal the composition and structure of the soil fungal community, analyze its diversity and functional characteristics, and reveal its relationship with soil nutrients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe three species preferred to grow on low-lying, ventilated and shaded negative terrain with good water drainage. There were significant differences in soil alkali-hydrolyzed nitrogen and available phosphorus among the three species. There were 336 fungal species detected in the samples. On average, there were different dominant groups in the soil fungal communities of the three species. The functional groups of soil fungi in the habitats were dominated by saprophytic fungi and ectomycorrhizae, with significant differences in diversity and structure. The co-occurrence network of habitat soil fungi was mainly positive. Soil pH significantly affected soil fungal diversity in the habitats of the three paphiopedilum species. The study and analysis confirmed that the dominant groups of soil fungi were significantly correlated with soil nutrients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe three species have similar habitat preferences, but there are significant differences in soil fungal composition, community structure and diversity. The functional group of fungi contains abundant saprophytic fungi, ectomycorrhizae, and a small amount of orchid mycorrhizae. The symbiotic relationships of the three species of soil fungi were harmonious, which was conducive to resistance to adverse environments. Soil environmental factors were significantly correlated with soil fungal communities, and pH significantly controlled fungal diversity. Our study on the habitat characteristics and soil fungal communities of the three wild \u003cem\u003epaphiopedilum \u003c/em\u003especies laid a foundation for future ex situ conservation and field return work.\u003c/p\u003e","manuscriptTitle":"Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 16:02:46","doi":"10.21203/rs.3.rs-4245399/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9392c2e-f97a-476c-bbb7-3a3dbbd04314","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-27T06:11:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-24 16:02:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4245399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4245399","identity":"rs-4245399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}