INTRODUCTION
The forest soil in the Andean highlands has high biodiversity and unique environmental characteristics. It is an endangered ecosystem, and little is known about its microbial composition. It is estimated that metagenomic DNA samples taken from undisturbed soil may contain 6,000 to 10,000 different microbial genomes (Torsvik & Øvreås 2002). Soil metagenomes can potentially reveal patterns of variation in the functionality of the soil biota and, from them, the general ecological patterns of community structure and ecosystem function (Fierer 2017).
With the exception of the some studies (Keating 1998; Thies et al. 2012), there is limited information on deforestation rates in South Ecuador, an important front of deforestation in the country. At the same time, this region is of particular interest and value for biodiversity conservation (Sierra et al. 2002), and to obtain valuable information about the microbiological components in this type of biomes (Tapia-Armijos et al. 2015).
Although many studies have shown that changes in environmental conditions are important drivers of changes in soil microbial diversity (Pausas & Bond 2020), this has been poorly documented in closed biome. There is reason to believe that the spatial diversity and richness of fungi among soil samples could save a close relationship when it is under a closed biome (Pausas & Bond 2020). The biome concept was first introduced to characterize structurally similar vegetation types in similar climates around the world. The biomes are representing distinct climate zones with unique characteristics (Moncrieff 2016). Closed biomes are forests, that is, tree-dominated ecosystems in which the density and leaf area is high enough to exclude shade- intolerant plants in the understory. Closed biomes typically have higher plant biomass than open biomes (Pausas & Bond 2020).
Fungi represent an essential functional component of terrestrial ecosystems as decomposers, mutualists, and pathogens, and they are one of the most diverse groups of Eukarya (Mueller et al. 2007). The study of the ecological factors that underlie the dynamics of fungal communities remains a challenge due to this high taxonomic and ecological diversity (Nordén et al. 2001; Genney et al. 2005; Koide et al. 2007). Moreover, microorganisms have a major impact on ecological restoration to recover soil functions although they have been rarely considered or incorporated into these restorative efforts (Donald et al. 2018).
The introduction of molecular biology and tools such as Sanger sequencing significantly increased the ability to characterize communities of microbes in situ (Malik et al. 2008; Stockinger et al. 2010). Recently, the advent of next-generation or high sequencing technology’s performance (NGS) have generated greater knowledge at an ever-faster pace (Lindahl et al. 2013; Van der Heijden et al. 2015). In the last decade, metagenomic analyses of microbial communities have become useful tools in the study of new microorganisms, genes, or biotechnological products (Steele & Streit 2005; Schmeisser et al. 2007; Steele et al. 2009), which are present in soil microbial communities without the need to cultivate them in vitro (Jansson et al. 2018).
The internal transcribed spacer region (ITS) is now widely used as a marker and DNA barcode validated for the identification of many species of fungi (Seifert 2009). With improvements in sequencing techniques and DNA databases recent studies have demonstrated the potential of high-performance ITS sequencing to quantify and characterize the fungal diversity of the soil (Kõljalg et al. 2005; Peay et al. 2016).
Recently, new methods have been developed that resolve amplicon sequence variants (ASVs) from Illumina-scale amplicon data without imposing the arbitrary dissimilarity thresholds that define molecular Operational Taxonomic Unit (OTUs) (Eren et al. 2013; Tikhonov et al. 2015; Eren et al. 2015; Callahan et al. 2016; Edgar 2016; Amir et al. 2017). ASV methods infer biological sequences in the sample prior to the introduction of amplification and sequencing errors and distinguish sequence variants differing by as little as one nucleotide. A similar class of methods developed for 454-scale data was typically used to ‘denoise’ sequencing data prior to constructing OTUs (Quince et al. 2011), while new ASV methods are explicitly intended to replace OTUs as the atomic unit of analysis. ASV methods have demonstrated sensitivity and specificity as good as or better than OTU methods and better discrimination of ecological patterns (Eren et al. 2013; Eren et al. 2015; Callahan et al. 2016; Needham et al. 2017).
In this article, we use high-throughput DNA sequencing to explore the diversity of soil fungi in high Andean forests of the closed biome, and observe the relationship with the altitudinal gradient, in three locations of the southern region of Ecuador, through the construction of metagenomic libraries, using Illumina technology, with an emphasis in microbiological soil restoration programs.