**1. Introduction**

Mountain ecosystems are rich in species diversity, and the climatic gradients are obvious within a relatively short distance, so it provides more possibilities for the research of biodiversity [1]. The diversity and community distribution of plants and animals are most frequently investigated in mountain ecosystems because of the environmental gradients and slant characteristics on a small spatial scale [2]. Peters et al. (2016) reported that the species richness of nearly half of the plant and animal taxa showed a decreasing trend with increasing altitude while the other half showed hump-shaped or bimodal distribution patterns in Mt. Kilimanjaro [3]. However, most of the current studies have focused on plants and animals, ignoring the interaction between soil microorganisms and plants [4]. In addition, soils are believed to be exceptionally biodiverse parts of ecosystems [5]. As widespread mutualists, fungi are symbiotic with plant roots and affect the growth and distribution of plants [6,7], and the effect may be different in different environment gradients [8], which play an important ecological role in ecosystem functions.

As the most widespread mutualists, arbuscular mycorrhizal (AM) fungi can form symbionts with 80% of plant species [9–11], which play important ecological functions in maintaining ecosystem balance in all kinds of ecosystems [11,12]. Research showed

**Citation:** Zhang, M.; Yang, M.; Shi, Z.; Gao, J.; Wang, X. Biodiversity and Variations of Arbuscular Mycorrhizal Fungi Associated with Roots along Elevations in Mt. Taibai of China. *Diversity* **2022**, *14*, 626. https://doi.org/10.3390/d14080626

Academic Editors: Michael Wink, Lin Zhang and Jinniu Wang

Received: 1 July 2022 Accepted: 4 August 2022 Published: 6 August 2022

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that AM fungi promote root growth and have positive effects on aboveground plant productivity through direct and indirect interactions [2]. Furthermore, AMF diversity was a key factor in maintaining plant biodiversity and ecosystem function [13–15]. Moreover, studying AMF biodiversity and distribution is the basis for predicting the evolution and succession of mountain ecosystems [16]. At present, more and more attention has been paid to the study of AMF diversity in mountain ecosystems. Yang et al. reported that elevations had a significant effect on AMF diversity and community distribution in the Qinghai-Tibet Plateau [17]. Gough et al. suggested that AMF are a ubiquitous group of soil microorganisms [18]. However, the measurement results of AMF diversity might be different using different methods in the same region. For example, Shi et al. (2014) researched AMF diversity and identified 63 AMF belonging to 12 genera by the traditional morphological identification method in Mt. Taibai [15], while Zhang et al. (2021) found 103 AMF species from soil samples, which belong to 19 genera using molecular identification method in Mt. Taibai [19]. It can be suggested that more AMF taxa are identified by the molecular method. Therefore, in this study, high-throughput sequencing molecular methods were used to explore AMF diversity and distribution in plant roots and investigate its influencing factors.

As the intersection of the flora of North China, Central China, and West China, Qinling Mountain is the natural dividing line between North and South China, with abundant species and resources. As the main peak of Qinling Mountain, Mt. Taibai is dominated by forest landscapes, rich in biological species, and is known as a green pearl in Western China [20]. The plant species is very rich in Mt. Taibai, which is one of the most abundant plant species in the temperate zone in China. Due to the different climatic gradients and the particularity of the vertical distribution of vegetation along altitudes, Mt. Taibai has become a natural place to study biodiversity [21].

Therefore, this study used molecular identification methods to explore the diversity and distribution mechanism of AMF associated with roots at different altitudes in Mt. Taibai, aiming to determine the biodiversity and variations of AMF with altitudes in the mountain ecosystem. It is expected to enrich the ecological theory of AMF by providing supporting data on different altitudes of mountain ecosystems.

### **2. Materials and Methods**

### *2.1. Description of Study Region*

This study was conducted in Mt. Taibai (23◦49 31–34◦08 11 N and 107◦41 23– 107◦51 40 E) of the Qinling Mountain, which lies in the ecological transition zone between the subtropical zone and the warm temperate zone and is an important east-west mountain range across the central part in China. As the main peak of the Qinling Mountains, Mt. Taibai is the first peak in the east of the Qinghai-Tibet Plateau in China, with the highest altitude of 3771.2 m. The climate zone of Mt. Taibai is obvious, which is divided into a temperate monsoon climate zone (800–1500 m), a cold temperate monsoon climate zone (1500–3000 m), a subarctic climate zone (3000–3350 m), and frigid climate zone (>3350 m). Besides, Mt. Taibai is rich in plant species and has a special geographic location, complex and diverse climate, and large altitude gradient, as well as one of the most abundant temperate plant species in China. In addition, the distribution of vegetation in the vertical zone of Mt. Taibai is also very special, which is of great significance to the study of the distribution of vegetation in the north and south of China and provides an ideal environment to conduct scientific research [22]. The plant distribution from the bottom to the top of Mt. Taibai can be divided into deciduous broad-leaved forest belt, coniferous forest belt, alpine shrub belt, and meadow belt. The deciduous broad-leaved forest belt is mainly distributed by *Quercus variorum* forest, *Quercus aliena* var. *Acuteserrata* forest and *Betula albo-sinensis* forest. The coniferous forest belt is mainly distributed by *Abies fabri* forest and *Larix gmelinii* forest. The alpine shrub belt and meadow belt are above 3350 m, mainly distributed dwarf creeping shrub, dwarf meadow, mossy community, and lichen community (Table S1). Meanwhile, it has always been a hotspot for biodiversity research.

### *2.2. Collection of Samples*

Twelve different altitudes were selected within the range of 663–3511 m in Mt. Taibai. At every target altitude, three 20 m × 20 m sample squares were set up, and the distance between each sample square was at least 50 m. In each sample square, a five-point sampling method was used to collect 0–30 cm soil, including all the plant roots and soil, and they were mixed as one sample. Three squares were considered three replicates. Finally, we separated all the mixed roots from the soil and put the mixed root samples and soil into different sealed bags, respectively.

Soil samples were used to determine the physical properties and nutrient elements after air-drying. All plant root samples were divided into the following three parts. The first part of each plant root sample was immediately stored in a −80 ◦C freezer for DNA extraction. The second part of each plant root sample was transported to the laboratory to carry out the determination of AMF colonization. The third part was stored in a 4 ◦C refrigerator to determine the nutrient elements, such as C, N, P, and C/N.

### *2.3. Bioinformatics Analysis of Sequence Data*

Genomic DNA was extracted from plant root samples using the Fast DNA SPIN Kit for Soil (MP Biomedicals LLC, Santa Ana, CA, USA) according to the manufacturer's protocols. The extracted DNA was subjected to nested PCR by a thermocycler PCR system (GeneAmp 9700, ABI, Foster City, CA, USA). PCR amplification was performed with primers AML1F (5 -ATCAACTTTCGATGGTAGG ATAGA-3 ) and AML2R (5 -GAACCCAAACACTTTGGTTTCC-3 ) by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA).

Purified barcoded amplicons were pooled in equimolar concentrations and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, CA, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Microbial community sequencing was conducted by Shanghai Majorbio Bio-pharm Technology using the Illumina-MiSeq sequencing platform. The data were analyzed on a free online platform (Majorbio I-Sanger Cloud Platform, available online: http://www.i-sanger.com, accessed on 3 August 2021). Used Uparse (version 7.1) software platform to perform taxonomic analysis of OTU representative sequences at a 97% similar level.
