The Diversity Analysis and Functional Prediction of Endophytic Fungi Community of Gymnadenia Conopsea from Different Altitudes in Tibet


 Gymnadenia conopsea has high economic value, and can be used as a medicinal and ornamental plant. Due to its low natural reproduction rate and overexploitation, the extinction of this plant is gradually accelerating. Understanding the composition and diversity of endophytic fungi is of great significance in promoting its propagation and the utilization of beneficial fungal strains. In this study, the diversity of fungal communities from roots, stems, leaves, fruits, and soils at four different elevations was studied with Illumina MiSeq sequencing. A total of 3,707,871 sequences were detected from all samples, and the number of clustering OTUs was 14,800. The OTUs were assigned to 4 phyla, 17 classes, 41 orders, 73 families, and 99 genera. The predominant fungal groups included Ascomycota and Basidiomycota, accounting for 33.71%-86.38% and 6.98%-58.30% of the total species, respectively. According to the alpha diversity index analysis, the diversity and richness of endophytic fungal communities in plant tissues at low altitudes were higher than those at high altitudes, while the diversity and richness of soil fungi were the opposite. In addition, principal coordinate analysis (PCoA) not only showed that the fungal community structure was correlated with altitude but also indicated tissue specificity of the community structure. Our study explored the composition of the endophytic fungal community among different tissues from different altitudes and included functional analysis, which might provide new ideas for saving the endangered species G. conopsea.


Introduction
Gymnadenia conopsea, which is widely distributed in Ireland, England, Russia, Japan, Nepal, the Korean Peninsula, and China, is a perennial and terrestrial herb of the Gymnadenia genus in the Orchidaceae family (Lin et al. 2020). In Tibet, G. conopsea mainly grows in forests, grasslands, and waterlogged meadows at altitudes of 1,265-4,700 m (Shang et al. 2017). Previous studies have shown that the tubers of this species have important antifatigue (Zhao and Liu 2011), antioxidative (Morikawa et al. 2006), sedative and hypnotic (Lin 2009), immunoregulatory (Li et al. 2006), antiaging (Si and Liu 2013), and antihyperlipidemic activities (Zhang et al. 2013). In recent years, tubers have also been used as an ingredient and tonic added to food by the local people in Tibet to strengthen their bodies and prevent illness (Shang et al. 2017). Due to overexploitation, habitat destruction, and its low natural reproductive capacity, the numbers of G. conopsea are decreasing rapidly (Shang et al. 2017;Lin et al. 2020; Gao et al. 2020). This species has been listed in the grade II section of endangered species by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (Lin et al. 2020). Therefore, it is urgent to protect the resources of this species.
Endophytes are a group of microorganisms with various morphologies, mainly including endophytic fungi and bacteria, that can enter into plants through the root cortex, wounds, or stomata and reside in different tissues or organs without causing any harm to the host plants ( The soil environment is different at different altitudes, and the abundance of the microbial community in soils is mainly affected by different nutrient levels (Ren et al. 2018). It has been reported that the fungal community structure varies signi cantly with altitude, mainly because of changes in soil nutrients (Siles and Margesin 2016). Yuan et al (2018) showed that endophytic fungal community diversity tended to decrease with increasing altitude. Previous literature reported different diversities of endophytes in the roots of G. conopsea from two geographically distant locations (Lin et al. 2020), but the interference of climate differences could not be well excluded. There are few reports on the effects of the same geographical location at different altitudes on the endophytic community structure in different tissues and rhizosphere soils of G. conopsea.
In this study, we aimed to investigate the diversity and composition of endophytic fungi of G. conopsea and rhizosphere soils at sites with different elevations and to further analysis their potential functions. Our results are expected to provide a basis for the conservation and arti cial cultivation of endangered species and provide new insights for the development and utilization of fungi that produce bioactive substances.

Sample collection and treatment
Tissue samples (roots, stems, leaves, and fruits) and rhizosphere soils of G. conopsea were collected from four sites in Nyingchi City, Tibet, China (Table 1). Three samples of each type were collected, for a total of 60 samples. The collected samples were randomly packed in sterile plastic bags and brought to the laboratory. All plant tissue samples were washed with running water and cut into 1-2 cm segments. Then, the samples were surface-sterilized with 75% ethanol for 30 s and 10% sodium hypochlorite for 5 min. The treated samples were washed repeatedly three times with sterile distilled water and dried on petri dishes. To test the sterilization procedure, the sterile water was incubated after the treatment to observe whether colonies were formed, and the absence of microorganisms indicated effective surface sterilization. Sequence processing and diversity analysis

Results
Richness and diversity analysis of the fungal community A total of 3,707,871 high-quality sequences from the stem, leaf, fruit, root, and soil samples collected from four sampling sites were obtained after read-quality ltering. The number of sequences in all samples of G. conopsea ranged from 47,235 to 68,027, with an average length of 237.5 bp ( Table 2). The obtained sequences were clustered into 14,800 OTUs at a 97% similarity level with the removal of low-abundance OTUs, ranging from 247 to 1,453.
All rarefaction curves showed that the number of OTUs reached saturation (99%), indicating su cient coverage of all fungal communities for further analysis ( Fig. 1). The alpha diversity indices, including Sobs, Chao, and Shannon indices, showed the richness and diversity of endophytes from all samples ( Table 2).
The Sobs index revealed that the richness of the fungal community in soils was the highest, followed by that in stems, and the lowest richness was in roots. Comparison of fungal community richness in four types of tissue samples from different sampling sites showed that the community richness from the FM location was the highest. The fungal richness in the stems and fruits collected at the LD sampling site was the lowest, while that in the leaves and roots of the LH site was the lowest. These results indicated that the community richness of endophytic fungi was affected by altitude, and the richness increased with decreasing altitude. According to the Shannon value, the highest diversity of the fungal community was found in soils, followed by stems, and the lowest diversity was found in roots. Among the four types of tissues, the diversity of endophytic fungi was highest at the FM site but lowest at the LD site, indicating that fungal diversity increased with decreasing altitude. Interestingly, the community diversity of soil fungi was higher at high altitudes (LH and LD) than at low altitudes (FM and LZ). The results implied that changes in the community richness and diversity of endophytic fungi and soil fungi were mainly governed by altitude. The plant tissues at the lowest altitude harbored the highest richness and diversity of the fungal community. According to the venn diagram ( Fig. 2), signi cant differences in OTUs were found in tissue and soil samples of G. conopsea. Among all samples, the number of OTUs was the highest in the soils, followed by the leaves, and the lowest in the roots, at 2719, 2260, and 1189 ( Fig. 2A). The richness of OTUs in stems (2073 OTUs) was higher than that in fruits (1286 OTUs). There were 252 shared OTUs in stems, leaves, and fruits. The number of OTUs in both roots and soils was 172, while that speci cally in roots and soils was 184 and 1325, respectively. The number of OTUs in tissue samples was the highest at the lowest altitude (FM site), but the results were different for soils, with the most OTUs at the high-altitude LH site. The lowest numbers of OTUs in fruits and stems were at the high-altitude LD site, and the lowest numbers in roots and leaves were at the LD site. The number of overlapping OTUs in fruits, roots, stems, leaves, and soils at the four sampling points was 101, 46, 140, 172, and 120, respectively. Our data implied that the amount of OTUs in plant tissues varied with altitude.

Analysis of endophytic fungi community composition
The sequences obtained from all samples were annotated and analyzed from the phylum to genus levels. In our study, the community composition of endophytic fungi varied among the different tissues and rhizosphere soils at different sampling locations. All the OTUs were analyzed at the phylum level, and 4 main phyla were predominant (Fig. 3A). Ascomycota was the dominant phylum with the highest relative abundance in all samples, accounting for 33.71%-86.38%, followed by Basidiomycota (6.98%-58.30%). The abundance of Ascomycota in fruits, leaves, and stems was signi cantly correlated with altitude, with high abundance at low altitude. In contrast, Basidiomycota in all samples were more abundant at higher elevations. Mortierellomycota existed only in roots and soils, with the highest relative abundance in the roots at the LZ collection site.
The sequences were classi ed into 73 families, which showed that the composition of the fungal community was different among different samples (Fig. 3B).
The relative abundances of Cladosporiaceae, Leptosphaeriaceae, Bulleribasidiaceae, and Helotiaceae were the highest in fruits, stems, leaves and roots, respectively. The most dominant group in fruits was Cladosporiaceae, occupying 20.77-31.87%, with the highest relative abundance at the lowest elevation, while this family was rare in stems and leaves and almost completely absent in soils. Leptosphaeriaceae was the predominant phylum of stems and showed a clear upward trend with elevation. Interestingly, the same phenomenon was observed in leaves but not in fruits. Bulleribasidiaceae predominated in the leaves, and the relative abundance ranged from 10.38-20.95%. The higher abundances of Bulleribasidiaceae at high altitudes were more pronounced in fruits and leaves than in stems. Helotiales_fam_incertae_sedis was the most dominant fungus in roots except at the high-altitude LD site, accounting for 18.33%-25.0%, while the abundance in stems and soils was less than 9%. It is worth noting that the relative abundance of Herpotrichiellaceae in the roots collected from the LD site reached the highest at 36.11%, while the relative abundance in other roots was lower than 8.43%. Six families of fungi only existed in the soils and roots, including Mortierellaceae, Russulaceae, Inocybaceae, Atheliaceae, Clavariaceae, and Leotiaceae, whose relative abundances were also inconsistent. The dominant fungal species found only in rhizosphere soils included members of the Archaeorhizomycetaceae, Thelephoraceae, Serendipitaceae, Hygrophoraceae, and Venturiaceae. The results illustrated that the abundance varied not only among the same types of tissues from different growth sites but also signi cantly varied among different tissue types. The abundances of communities between the rhizosphere soils and roots were different at the family level, but there were similarities in composition.
To further reveal the abundance and structure of the fungal community across the plant tissues and soils at the genus level, we performed heatmap analysis, to elucidate the distribution of the top 50 most abundant genera in the endophytic communities. As shown in Fig. 4, the distribution and structure of fungal communities were different among different sample types. For example, the fungal abundance in the roots and soils differed at the genus level. The distribution and abundance of several fungi, including Cladosporium, Rachicladosporium, Protomyces, Vishniacozyma, and Heterocephalacria, in the stems, leaves, and roots were higher than those in the fruits and soils. In addition, the abundance of Archaeorhizomyces varied among different types of samples, with the highest abundance in soils and increasing with elevation. Cadophora had the highest abundance in roots, followed by stems, and the abundance varied greatly at different altitudes. We observed from the heatmap that the fungal structure from different sample types, such as soils and fruits, was signi cantly different, while the same types had similar community structures. Compared with sample type, altitude had less effect on fungal community composition.

Comparative analysis of endophytic fungal communities at different elevations
To explore differences or similarities in the microbial community composition between samples, principal coordinate (PCoA) analysis based on Bray-Curtis distances was used to show that the between-sample distance re ected a similar composition between microbial communities at the OTU level (Fig. 5). The closer the samples were, the more similar the species composition was. In Fig. 5A, the roots and rhizosphere soils were grouped together and more concentrated, which implied that their community structures were similar. The plant tissues (leaves, fruits, and stems) formed separate clusters that were relatively dispersed. The results suggested that there were similar community structures among the roots and soils, which obviously differed from those of the other tissues. In addition to roots, the samples from plant tissues at the FM and LZ sites at low altitudes were more aggregated than those at the high altitude sits of LH and LD, which implied that community compositions were similar. The results indicated that the community structure was not only related to the sample type but also to altitude. This was also demonstrated in Fig. 5B. The points of the samples from the LH and LD sites were closer than those of the other sites, indicating that they were more similar in community structure. The distances of the fungal communities at the FM and LZ sites were closer, and the species compositions were more similar. The similarity of fungal groups might have relatively strong linkages to altitude.
In uence of soil characteristics on the fungal community structure in different samples The soil samples collected from four sites were measured, and 8 indices of soil characteristics were determined, including total phosphorus (TP), total potassium (TK), total nitrogen (TN), available nitrogen (AN), pH, electrical conductivity (EC), available phosphorus (AP), and soil organic matter (SOM) ( Table 3). Redundancy analysis (RDA) was used to explore the relationship between soil environmental factors and microbial community structure in all samples (Fig. 6A).  To further evaluate the correlation between the fungal community composition and the environmental factors, we analyzed the correlation heatmap using the Spearman correlation coe cients at the genus level (Fig. 6B). The six environmental factors, including TP, EC, TK, pH, and SOM, exerted a more signi cant impact on the microbial community structure than AP and TN, which was consistent with the RDA results. For the top 50 most abundant genera of the fungal communities, unclassi ed_o_Chaetothyriales, Trichomerium, Russula, unclassi ed_c_Eurotiomycetes, Cladophialophora, unclassi ed_f_Venturiaceae, Mrakia, Knu a, Cladophialophora, Ceratobasidium, and unclassi ed_o_Chaetothyriales were positively related to the total P content, while unclassi ed_f_Didymellaceae, unclassi ed_f_Hyaloscyphaceae, unclassi ed_c_Leotiomycetes, and Protomyces were negatively correlated. Interestingly, unclassi ed_f_Chaetothyriaceae and Trichomerium were negatively correlated with TK and pH. In addition, unclassi ed_c_Eurotiomycetes was positively correlated with TP but negatively correlated with EC, AN, TN, SOM, and TK, with signi cant differences. There were 7 species positively related to pH with signi cant differences, among which the P value of Inocybe was less than 0.001. Both pH and TP were positively correlated with unclassi ed_f_Venturiaceae.
Leucosporidium and Tetracladium were positively correlated with pH and EC. The correlation analysis veri ed that the differences in microbial structure were signi cant among the different soil environmental factors.
Predicted functional pro les of the metagenome The abundance of the MetaCyc pathway was predicted using PICRUSt2 (Table S1). A total of 73 pathways were identi ed. Functional gene families were mainly involved in the circulation of carbon and the metabolism of amino acids, fatty acids, and nucleotides. Among these genes, the relative abundances of those related to aerobic respiration, fatty acid oxidation, the glyoxylate cycle, GDP-mannose biosynthesis, the pentose phosphate pathway, myoinositol biosynthesis, pyruvate fermentation, methyl ketone biosynthesis, and guanosine nucleotide degradation were markedly high. In addition, genes with several functions associated with palmitate biosynthesis I, sulfate reduction, and the superpathway of ubiquinol-6 biosynthesis were more abundant in soils and roots than in other tissues.

Discussion
In this study, we investigated the fungal community structure in different plant tissues and rhizosphere soils of G. conopsea sampled from four sites in Tabet through high-throughput sequencing to explore the relationship between endophytic fungi and plants from a deeper perspective, with the aim of exploring the role of endophytic fungi and laying the foundation for the development and utilization of endophytic fungal resources.
A total of 14,800 OTUs belonging to 4 phyla, 73 families, and 99 genera obtained from tissues and rhizosphere soils were analyzed. It was previously reported that mycorrhizal fungi associated with orchids were mainly Basidiomycota and Ascomycota (Sisti et al. 2019). In our study, Ascomycota (33.71%-86.38%) was the dominant phylum, with the highest relative abundance in all samples, followed by Basidiomycota (6.98%-58.30%), while Mortierellomycota had the lowest relative abundance and existed only in roots and soil. These ndings were similar to previous studies by Lin et al. (2020), who explored the differences in fungal communities from the roots and soils of G. conopsea. We also found that the community abundances of endophytes and soil fungi were different at the four sampling sites. The abundance of Ascomycota in fruits, leaves, and stems at low altitudes was higher than that at high altitudes, while the abundance in roots and soils was not consistent. Interestingly, the abundance of Basidiomycota was the opposite, with higher abundance at higher altitudes. In the current study, Cladosporium and Cadophora were the dominant species, with different degrees of richness in different tissue samples. Among them, Cladosporium was the dominant genus in fruits, accounting for a relative abundance of 10.11%-21.96%, and the abundance in stems was less than 5%; this genus was barely detected in other samples. These results imply that this species plays an important role in fruit development of G. conopsea. The abundance of Cadophora was the highest in roots, and this genus was con rmed by Berthelot et al (2016) to enhance plant growth. Ceratobasidium also had the highest abundance in roots. Ercole et al (2015) observed mycorrhizal fungi such as Ceratobasidium with diverse communities in the roots and protocorms of photoautotrophic orchids. N uptake from inorganic sources and transport to the protocorm by the genus Ceratobasidium were revealed by Kuga et al. (2014). However, the abundances of Phialophora and Aspergillus were only 1.22%-5.25% in roots, but that of Gibberella was less than 5% and 1% in stems and leaves, respectively, which might also contribute to the extinction of G. conopsea. The symbiotic relationship between orchid plants and endophytes exerts a signi cant in uence on the growth and natural propagation of orchids. In other words,  (2020) also showed that the diversity of endophytes in the roots of orchids was related to the soil environment. RDA indicated that the soil environment exerted a signi cant in uence on the fungal community. In this study, the low abundance of fungal communities associated with plant growth promotion might be affected by the soil environment, which may be one of the reasons for the endangerment of G. conopsea in Tibet.
Endophytes can not only promote plant growth but also produce bioactive components (Sisti et al. 2019 contained a variety of protective genes that helped the fungus survive in adverse environments. We also found that the abundance of Herpotrichiellaceae in roots was high, with the highest at the LD collection site; this family may aid G. conopsea adaptation to the environment of the Qinghai-Tibet Plateau, which is characterized by low temperature, strong UV ultraviolet light and low oxygen. Several species of Nectriaceae have previously been reported to have potential as biocontrol agents and biodegraders in industrial applications (Lombard et al. 2015;Ye et al. 2020). Similarly, the high abundance of these species in roots and rhizosphere soils in our results might make the plant more adaptable to the soil environment. In addition, secondary metabolites such as mycotoxins or antibiotics were secreted by several species belonging to the family Trichomeriaceae results also showed a high abundance of amino acids and carbohydrate metabolism. Amino acid metabolism mainly involved leucine, tyrosine, tryptophan, serine, valine, glycine, methionine, threonine, and arginine. Jiang et al (2018) isolated 9 kinds of amino acids from G. conopsea, which have high nutritional and medicinal value because they could maintain nitrogen balance in the body. Yu (2017) found that polysaccharides had the effect of delaying aging and alleviating fatigue through study of the pharmacological activity of polysaccharides in G. conopsea. Therefore, the metabolism of amino acids and carbohydrates in endophytes might be bene cial for improving the nutritional and medicinal value of G. conopsea. The natural propagation rate of G. conopsea is low, arti cial cultivation of this species is di cult, and human destruction of habitat is increasing, which will cause the population to decrease. To avoid the decline of G. conopsea caused by the exploitation and utilization of the species for medicinal resources, effective medicinal ingredients can be obtained from microorganisms by mass culture in the future. Our study aimed to analyze the fungal community diversity and composition of G. conopsea, which may be valuable for screening fungi with biological activity.