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.