Characterizing Bacterial Communities in Agroecosystems of the UNESCO Global Geopark Mixteca Alta, Oaxaca

Abstract

This study explores the diversity and functions of microbiomes in ancient agroecosystems of the Mixteca Alta Geopark (MAG). Microbiome analysis could provide insights into soil bacterial communities and their role in enhancing soil fertility, nutrient cycling, and plant growth. We used 16S rRNA gene amplicon sequencing to identify key features in the composition of the microbiota of the Lama-bordo, Valley, and Terrace agroecosystems in MAG. Analysis of agroecosystem soils revealed 21 bacterial phyla, with Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi dominating. These microbial communities contribute to soil health, carbon cycling, and disease suppression. The study identified specific phylogroups and metabolic pathways associated with nutrient-rich environments like Lama-bordo and Valley, and nutrient-poor, sandy soils like Terrace. Soils from Lama-bordo and Valley were grouped due to microbiome similarity despite geographic separation, whereas Terrace soils differed. Nutrient-rich Lama-bordo and Valley soils host copiotrophic bacteria, while nutrient-poor Terrace soils favor oligotrophic species like Acidobacteria. Functional analysis of microbiomes reveals distinct metabolic pathways, including antibiotic biosynthesis (streptomycin, vancomycin) suggesting a role in plant disease resistance, amino acid pathways indicating active nitrogen cycling, and vitamin B5 and lipoic acid pathways contributing to energy metabolism and antioxidant functions.

1. Introduction

The Mixteca Alta is a region characterized by mountains and valleys located in the northwest of the state of Oaxaca, at the convergence of the Sierra Madre del Sur and the Sierra Madre Oriental [1]. This region features geological formations from the Cretaceous, Tertiary, and Quaternary periods. The predominant climates are temperate humid and subhumid, with Cambisols, Leptosols, Luvisols, Regosols and Vertisols being the main soil types [2]. The major vegetation types in the region include pine–oak mixed forests, xeric scrub and grassland. Land use is primarily associated with rainfed agriculture and induced grasslands, primarily developing in the foothills and valleys [3]. Due to its geodiversity, the Mixteca Alta Geopark (MAG) was established in this region as part of the UNESCO World Geoparks program. The MAG, influenced by erosion, human activity, and distinctive geological features, serves as a field laboratory for studying the processes of human occupation and natural resource use that have contributed to erosion, shaping the current geological landscapes [4].

The farming systems, particularly lama-bordo terraces in the Mixteca Alta of Oaxaca, have been present in the region for more than 3400 years [5]. There are very old records of agricultural activities in the area where MAG is located. These activities, associated with agricultural production systems, have shaped the geographic landscape of the region and can be observed in the erosion processes that are still present to this day [1]. The Mixtecs, who lived in this area, developed agricultural techniques that allowed them to survive in these variable climatic conditions. The first places that were permanently used for agriculture in the Mixteca region were the alluvial plains. Later, they utilized the foothills and finally constructed lama-bordos. The latter were built perpendicularly along the course of water currents [6]. They also constructed terraces on the slopes of mountains that made it possible to cultivate on steep slopes while avoiding soil erosion and nutrient loss [7].

In recent years, the study of agricultural systems in the MAG has been approached from the perspective of the diversity of domesticated and cultivated plants, the agricultural systems implemented in the region, as well as the cultural processes that arise around agricultural practices [8,9,10,11,12,13]. However, a still unknown aspect of the agroecosystems present in the MAG is the bacterial communities that support both the soil and crops grown there. The complex network of biotic interactions between crops and soil microorganisms—such as bacteria, fungi, archaea, and viruses—can benefit plants by optimizing nutrient uptake, promoting growth, and combating pests [14]. Bacterial communities, integral to soil health, play a crucial role in agroecosystems, influencing soil fertility, crop productivity, stress tolerance, soil aggregate formation, weed suppression, moisture retention, and erosion control [15,16,17,18,19]. The interaction between soil bacteria and plants in agroecosystems is dynamic, shaped by plant hosts and land management practices. Studies have shown that microbial community assemblages are species-specific and vary with different crops, forming distinct microbial communities. These assemblages also shift throughout the growing season, responding to developmental stages [20,21,22]. Agricultural practices can alter soil structure, impacting microbial processes at a microscale. Such changes can have significant consequences, including soil erosion, decreased fertility, and increased greenhouse gas emissions [23,24,25]. Tillage, for example, disrupts the soil microbiome and its ecological functions. Research indicates that no-till soils exhibit a greater microbial capacity for nitrogen mineralization and higher bacterial diversity compared to plough-tillage [26]. Therefore, a two-way interaction exists in agroecosystems where bacterial communities contribute to the modification of the soil and ecological interactions of these agroecosystems, while agricultural practices carried out by local farming communities impact microbial processes. These processes remain unknown in MAG agroecosystems and have not been studied to date.

This study investigates the structure and diversity of microbiomes in agroecosystems within the Mixteca Alta Geopark (MAG) of Oaxaca. It focuses on the impact of different agricultural practices on bacterial communities in three agroecosystem types—lama-bordos, terraces, and valleys—that have been cultivated for over 3400 years [5]. The research explores how these traditional land-use practices shape different bacterial compositions, with variations in soil microbiome diversity and function reflecting differences in management and environmental factors. To achieve this, 16S ribosomal gene sequencing of bacterial communities was conducted across the three agricultural sites currently used for cultivation. Through genomic analysis, we have begun to identify and describe the bacterial species that make up the microbiomes associated with these agroecosystems. Additionally, we evaluated the abundance and general composition of the bacterial communities as well as their metabolic processes within each agroecosystem. To date, this is the first report on the microbiomes present in these particular agroecosystems within the Mixteca Alta Geopark, and the first study on microbiomes in traditional agroecosystems in Oaxaca.

2. Materials and Methods

2.1. Soil Sampling

To describe the bacterial communities found in the soil of the agroecosystems of the Mixteca Alta Geopark in Oaxaca, the sites of Valley (17°30′14″ N 97°20′33″ W, 2105 MASL), Lama-bordo (17°35′34″ N 97°21′51″ W, 2523 MASL) and Contour Terraces (17°35′44″ N 97°22′5″ W, 2560 MASL) were included (Figure 1). These sites were chosen because they represent the most important agroecosystems that have been developed in the MAG by local farming communities over a long period. At each location, four soil samples were collected from a one-meter square area, with one sample taken at the center and three from the edges. Two grams of soil were extracted from a depth of 20 cm, mixed with 6 mL of LifeGuard Soil Preservation buffer (Qiagen, Germantown, MD, USA), and then stored at −20 °C for preservation. The samples were taken in May 2023, during the dry season.

2.2. DNA Extraction and Sequencing

DNA extraction, library preparation, and 16S rRNA hypervariable regions V3–V4 sequencing were conducted by the Research and Testing Laboratory Genomics (Lubbock, TX, USA) as previously described [27]. Briefly, DNA was extracted using the Qiagen MagAttract PowerSoil DNA KF Kit (Qiagen, Germantown, MD, USA). The V3–V4 regions of the 16S rRNA gene were amplified with the primer pair 357wF (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′). PCR reactions were performed on an ABI Veriti thermocycler (Applied Biosystems, Carlsbad, CA, USA). Amplified products were checked, pooled, and size-selected twice using SPRIselect Reagent (Beckman Coulter, Indianapolis, IN, USA). Quantification was performed with a Qubit 4 Fluorometer (Life Technologies, Grand Island, NY, USA). Paired read (2 × 250) sequencing was performed using an Illumina MiSeq (Illumina, San Diego, CA, USA).

2.3. Data Analysis

Sequencing data were analyzed using the Quantitative Insights into Microbial Ecology 2 v2021.2 (QIIME 2) pipeline [28]. At the Lama-bordos study site, only three samples were analyzed, as one was excluded due to significantly lower richness compared to the other three (Supplementary Figure S1). Sequencing errors were corrected using the DADA2 [29] plugin in QIIME 2. Taxonomic classification was performed with a Naive Bayes classifier trained using the SILVA v132 database [30] and the specified primers. Taxonomic assignment was conducted with the “q2-feature-classifier” plugin under default settings. A phylogenetic tree was constructed using the “phylogeny” plugin in QIIME 2. Rarefaction curves and various alpha diversity indices, such as observed features, Chao estimator and Shannon index, were computed using the R package phyloseq [31]. Additionally, beta diversity was assessed using Double Principal Coordinate Analysis (DPCoA) [32]. Functional profiles of bacterial communities were inferred using PICRUSt2 v2.5.2 [33,34], based on 16S rRNA data processed with QIIME 2. KEGG [35] orthology (KO) numbers were used to estimate pathway abundance. Statistical analysis of the relative proportions of identified taxa and their predicted functions was conducted using STAMP software version 2.1.3 [36] to determine significant differences. White’s non-parametric two-sided t-test was applied for hypothesis testing to evaluate these variations. The confidence interval was calculated using the difference between the mean proportions (DP) method with bootstrap. The thresholds for p-value and effect size were 0.05 and 1.0, respectively. Results with p-value < 0.05 were considered significant for both taxonomic and metabolic data.

3. Results

3.1. Diversity and Composition of Bacterial Communities of Agroecosystems of GMA

The taxonomic assignment of bacterial communities was based on 16S rRNA gene amplicon sequencing, analyzed with QIIME 2 [28] software and the Silva RNA database [30]. Almost all reads across the three sites were classified under the Bacteria domain. In contrast, the Archaea domain showed minimal representation, accounting for only 0.20% in one replicate from the Valley site. Phylogenetic assignments at 99% similarity identified 21 phyla, including 19 with known cultivable representatives, such as Acidobacteria, Proteobacteria, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes. Meanwhile, two phyla WPS-2 and BRC1, do not have a cultivable representative thus far. They have a minimal representation in Lama-bordos with 2.5% and Valley with 0.05%, respectively. Few sequences were not assigned to any taxonomical group and were only identified as Bacteria, with 0.385% of sequences in the Valley site, 0.947% in Lama-bordos, and 1.493% in Terraces. The relative abundances of microbial taxa at the phylum level showed that about 80% of bacterial sequences were assigned to Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi (Figure 2). The phylum Acidobacteria presents a higher abundance in the samples taken at the Terraces site, while Proteobacteria present their highest abundance at Lama-bordos and Valley sites. Two phyla are present in a differential manner: Bacteroidetes is only found in Lama-bordos and Valle sites; and WPS-2, currently known as Eremiobacterota, is only present at the Terrace site. In addition, 47 classes, 113 orders, 176 families, and 148 genera were identified (Supplementary Table S1). At the family level, the most abundant in the three sites were Solibacteraceae, Gemmatimonadaceae, Xanthobacteraceae, Pyrinomonadaceae, Sphingomonadaceae, Gemmataceae, Pseudonocardiaceae, and Geodermatophilaceae (Supplementary Figure S1). Of the previous families, Solibacteraceae exhibits the highest abundance in the Terraces samples, with a relative abundance of 17.2%. In the other two sites, the abundance of this family reached a maximum of 8.9% at the Lama-bordos sites. While Gemmatimonadaceae had a high abundance in the samples obtained from Lama-bordos and Valley sites, with 7.3% and 6.2%, respectively. In contrast, Terraces had an abundance of 2.1%.

3.2. Diversity Estimates

To evaluate the diversity of the microbiomes present in the analyzed locations, the alpha diversity was estimated in the three agroecosystems (Figure 3). According to the Chao 1 estimator, the diversity analysis of the samples from Lama-bordo locations showed a higher value than that of Terraces and Valley sites. The Shannon index diversity in the Lama-bordo site was between 5.4 and 5.5, indicating a balanced distribution of bacterial communities. In contrast, samples from the Terrace site showed a low Shannon diversity value, with values between 4.4 and 4.8. Rarefaction analysis was conducted based on phylogenetic annotation of reads using the phyloseq package. The rarefaction curves generated at the species level (Supplementary Figure S2) revealed that all samples reached a plateau, indicating that the sequencing depth was adequate for a comprehensive description of the bacterial diversity in the soils of the agroecosystems.

We next attempted to determine how similar are the samples obtained from the different sites in terms of their identified microbial communities. To achieve this, we employed Double Principal Coordinates Analysis (DPCoA) [32] to examine the differences among species using a dissimilarity matrix and the species distribution among communities using an abundance/absence matrix (Figure 4). It can be seen that the samples from Lama-bordo and Valley are clustered together to the left, on axis 2; while the Terrace site is separated on the right. Changes in physico-chemical variables such as temperature, salinity or agricultural practices that occur throughout the year seem to have an impact on the conformation of the structure of microbial communities in agroecosystems.

3.3. Agroecosystem–Taxonomy Association

To evaluate potential associations between the identified taxonomic groups and the agroecosystems from which they were sampled, statistical analysis was conducted using the STAMP software v2.1.3 [36]. This analysis was performed at both the phylum and family taxonomic levels and compared relative abundances between Lama-bordo (n = 3), Valley (n = 4) and Terrace (n = 4) sites. From this analysis, we found that the Planctomycetes phylum was overrepresented in the Valley location, while the Gemmatimonadete phylum was shared with Lama-bordo site. In the Lama-bordo site, the Proteobacteria phylum is enriched compared to the other two agroecosystems. Finally, we observed an overrepresentation of Acidobacteria and WPS-2 (Eremiobacterota) at the Terrace site along with Actinobacteria (Figure 5A–C). Analysis at the family level reveals a large proportion of Solibacteraceae, Acetobacteraceae, Acidobacteriaceae, Acidothermaceae and Ktedonobacteraceae in the Terrace site, while the Geodermatophilaceae family is shared with the Valley site. In the Valley site, Beijerinckiaceae, Rubrobacteriaceae, Gemmataceae, Azospirillaceae, WD2101 soil group, and Micromonosporaceae families are overrepresented; whereas Pyrinomonadaceae and Gemmatimonadaceae were shared with Lama-bordo site. Lastly, in the Lama-bordo site, Xanthobacteraceae, and Pirellulaceae families have a large proportion (Figure 5D–F).

3.4. Functional Profile Prediction of Agroecosystems

To assess the potential molecular functions of the prokaryotic communities identified in the agroecosystems of GMA, we conducted a prediction of their metabolic capabilities. We utilized PICRUSt2 v2.5.2 software together with the KEGG database to perform functional annotation at level 3, associating specific pathways with particular functions. We can group the major metabolic pathways (relative abundance > 1.5%) found in the agroecosystems into antibiotics production (biosynthesis of ansamycins, vancomycin, streptomycin), amino acid synthesis (glutamine, glutamate, valine, leucine, isoleucine), and metabolism of cofactors and vitamins (pantothenate, lipoic acid) (Figure 6).

To identify statistically significant pathways, we employed STAMP and considered molecular functions with a p-value < 0.05 as significant. For this purpose, we conducted a similar analysis to that which was performed for the evaluation of the environment-taxonomy association. Lipopolysaccharide biosynthesis, lipoic acid, and fatty acid metabolism were significantly present in Lama-bordo; while flagellar assembly and D-arginine and D-ornithine metabolism were shared with the Terrace site. In this latter agroecosystem, starch and sucrose, galactose, glycosaminoglycan degradation, and bacterial chemotaxis were dominant. Finally, in the Valley agroecosystem, Geraniol degradation, valine, leucine, and isoleucine biosynthesis, as well as pantothenate and CoA biosynthesis, were predominant (Figure 7).

4. Discussion

4.1. Microbiomes of Ancient Agroecosystems of Mixteca Alta Geopark

Soil bacterial communities are known to improve soil fertility, increase carbon storage, combat crop pathogens, enhance nutrient availability, and promote plant growth [19,37]. In recent years, research on agroecosystem microbiomes has increased due to their key role in ecosystem resilience and functioning [23,38,39,40]. The analysis of bacterial diversity and community composition in the agroecosystems of the Mixteca Alta Geopark offers valuable insights into the microbial ecology of these environments and their potential functions within the studied agroecosystems. Our results identified 21 bacterial phyla across the sites, most of which have known cultivable representatives. The presence of these phyla suggests that the bacterial communities in these agroecosystems are diverse. This high diversity of bacterial phyla indicates a potentially resilient microbial community capable of supporting various ecological functions, such as soil health and fertility. The phyla Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi dominate the microbial communities in the three agroecosystem sites of GMA, comprising about 80% of the bacterial sequences. This is in agreement with other reports, suggesting that these ecosystems share common bacterial community structures with other agroecosystems [41], despite the unique environmental conditions of the Mixteca Alta. Additionally, these four phyla have been reported to be part of the maize microbiome [42,43] due to their functional roles in soil processes, including nutrient cycling, organic matter decomposition, and plant interactions [44,45]. Actinobacteria and Entotheonellaeota (the latter present exclusively in the Valley agroecosystem) have been reported as potential biomarkers associated with intercropping systems, such as corn/beans [46]. Actinobacteria, in particular, have been linked to alkaline soils such as those in the MAG region, rich in organic matter, with favorable chemical properties, including high levels of C, N, K, Mg, and Na. They contribute significantly to the carbon cycle, suppress plant diseases, and promote plant growth [47,48]. It is now known that agroecosystems have their own distinct microbiomes; depending on the crop and soil management practices, the composition of the bacterial community in the soil can vary significantly. For example, Bulgarelli et al. [49] showed that the Firmicutes and Chloroflexi phyla were more abundant in the bulk soil of barley crops, while Xiong et al. [50] found that the bacterial family Chitinophagaceae (Bacteroidota phylum) was more abundant in the soil of a wheat/barley rotation agroecosystem. In the maize cultivation sites analyzed in this study, Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi appear to be the representative phyla of the GMA agroecosystems.

At the family level, Solibacteraceae, Gemmatimonadaceae, Xanthobacteraceae, Pyrinomonadaceae, Sphingomonadaceae, Gemmataceae, and Acetobacteraceae were the most abundant. Our findings are consistent with those of Górska et al. [48], who reported the presence of the families Solibacteraceae, Sphingomonadaceae, and Gemmatimonadaceae in soils subjected to plow tillage, Ca, N, P, K and manure fertilization. The presence of Solibacteraceae in soils has been reported to be associated with plant protection against certain fungal pathogens, such as Fusarium oxysporum, and their role in the soil’s biogeochemical carbon cycle [51]. Additionally, their increased abundance has been linked to the use of manure in soils [52]. Similarly, members of the Sphingomonadaceae family are known to be antagonistic to plant pathogens and promote plant growth. Several species within this family can degrade xenobiotic and recalcitrant (poly)aromatic compounds of natural or anthropogenic origin [53]. It has been reported that the relative abundance of the Xanthobacteraceae and Pyrinomonadaceae families increases in soils following the addition of organic amendments, with a positive correlation observed between their presence in these soils [54].

4.2. Ecological Adaptations in Agroecosystems of Mixteca Alta Geopark

With the aim of identifying phylogroups that were overrepresented in the agroecosystems analyzed here, we applied a statistical analysis of the relative abundance in the sampled sites. Our results showed that the Lama-bordo and Valley agroecosystems were statistically abundant in copiotrophic-associated phyla, such as Planctomycetes, Gemmatimonadetes, and Proteobacteria [41] (Figure 5A–C). Copiotrophs, or eutrophs, are organisms associated with nutrient-rich environments and are generally adapted to rapidly utilizing resources when available. They grow and reproduce quickly in the presence of abundant nutrients, particularly in carbon-rich soils, and flourish in environments with high carbon mineralization rates [55,56] such as Lama-bordo and Valley sites, which are documented as nutrient-rich soils [9]. Lama-bordos are rich in organic matter, with deep, wet soils. Their construction is intended to collect soil from the hillsides and from the sediment carried by water currents that form during the rainy season. In the case of Valley, farmers describe the soils as black, deep, and of good quality. In both cases, Lama-bordos and Valley agroecosystems are rich in organic matter with deep soils [9]. Lama-bordos, influenced by their parent material, exhibit better soil quality indicators compared to nearby agricultural lands and show soil quality similar to high-quality fallow lands, though there is variation across different lama-bordo systems [57,58]. Planctomycetes are known to have a significant impact on soil ecosystems by decomposing organic matter and sequestering carbon, making them critical to the carbon and nitrogen cycles. They exhibit flagellar motility and use chemotaxis to reach nutrients in nutrient-rich environments [59,60,61]. They are also associated with high levels of dissolved organic carbon, dissolved organic matter, and carbohydrate metabolism [62,63]. Gemmatimonadetes are abundant in agricultural soils with neutral to alkaline pH, such as the calcareous soils found in the Geopark, and are frequently associated with plants and the rhizosphere. Members of this phylum possess the metabolic potential for the reduction in N₂O, one of the strongest greenhouse gases [64]. Their ability to harvest light provides them with additional energy and improves the efficiency of carbon utilization. Gemmatimonadetes are suggested to be adapted to dry environments, as they occur in high relative proportions in semiarid and arid soils and deserts. This phylum has also been found to be positively correlated with vegetation restoration and with total carbon, nitrogen, and phosphorus in the soil [65]. Together with Gemmatimonadetes, the Proteobacteria phylum is of great importance to global carbon, nitrogen, and sulfur cycling [66]. Proteobacteria provides carbon-rich conditions that support high metabolic activity, rapid growth, and the propagation of plants; also, they are usually associated with habitats with high NO₃⁻-N and enriched nutrients [67,68]. At the Terraces site, the oligotrophic phylum Acidobacteria is particularly abundant (Figure 5A–C). Terraces are built on the slopes of the Geopark hills, where the land has gradients greater than 10%. The soils are thin, sandy, and dry [9]. This phylum has been associated with habitats that are relatively nutrient-poor [59]. De Castro et al. [69] reported the presence of the Acidobacteria phylum in nutrient-poor, aluminum-rich soils with low phosphorus content. One feature of Acidobacteria is their exopolysaccharide (EPS) production. The functions of EPS in soil are numerous; it may be involved in the formation of the soil matrix, serve as a trap for water and nutrients, and facilitate bacterial adhesion, which can promote soil aggregate formation [70]. This is fundamental in thin, nutrient-poor soils, such as those at the Terraces site, which have already been documented as nutrient-poor [9].

The varying environmental conditions of the MAG agroecosystems have led to a differential association of phyla with distinct ecological functions. In the Lama-bordos and Valley agroecosystems, copiotrophic organisms are statistically abundant, whereas oligotrophic phyla predominate in the Valley. This is also evident in the DPCoA analysis, where samples from Lama-bordos and Valley are grouped together and separated from those of the Terrace site (Figure 4). These results reveal a distinct composition of the microbiomes present in the MAG agroecosystems, both in terms of species and abundance, reflecting the different soil conditions. Our study shows that the microbiomes of the Lama-bordos are more similar to the Valley soils, despite being geographically distant (Figure 1). The nutrient-rich environments of the soils of Lama-bordos and Valley sites influenced the richness of bacterial species that inhabit the soils of these agroecosystems. As we can observe in Figure 3, the alpha diversity of Lama-bordo and Valley is greater than the value obtained from the Terrace agroecosystem. From an ecological perspective, the individual species present in the soil are less important than the overall functionality that the entire microbial community provides to soil health and resilience. High functional microbial capacity generally correlates with greater species diversity [71]. The relatively nutrient-poor soil of Terrace has as a result a minor value of alpha diversity.

It is worth noting that some phylogroups, although not statistically significant in their abundance, are biologically important due to the ecological and molecular functions they perform in soils. Examples include the Bacteroidetes and Nitrospirae phyla (Supplementary Table S1), which are present only at the Lama-bordo and Valley sites. Bacteroidetes have been reported as one of the abundant phyla in metagenomic studies of soils [71], but their abundance was not statistically significant in the agroecosystems of MAG. Bacteroidetes were present in only low proportions at the Lama-bordo and Valley sites. Despite their low abundance, Bacteroidetes hold great ecological importance, as they degrade the vast majority of complex glycans available in soils, which come from growing plants, leaves, stems, and other recently dead plant tissues [72]. Additionally, they have the potential to solubilize organic phosphorus, making it available to plants [73]. The Nitrospirae phylum, on the other hand, participates in the nitrogen cycle through the process of nitrification. Nitrification is a crucial process for producing nitrate, a key nitrogen source for soil plants. It involves two independent steps: ammonia oxidation (NH₃ to NO₂⁻) and nitrite oxidation (NO₂⁻ to NO₃⁻) [74]. Members of the phylum Nitrospirae, present only at the Lama-bordo site, have the potential to oxidize both ammonia and nitrite, thereby contributing to soil nutrients. Although their abundance is low, their ecological importance in providing nutrients to plants is significant [75]. Another example of a biologically important phylum is WPS-2 (Eremiobacterota), which is present in low abundance only at the Terrace sites. Members of WPS-2 have been reported as significantly more abundant in bare soils than in vegetated soils and are adapted to extreme dryness, high radiation, and very limited organic matter [76]. The WPS-2 phylum is involved in breaking down plant metabolites and aromatic compounds, such as glycolate, which is a potential substrate. A key trait of this group is their ability to perform atmospheric chemosynthesis by oxidizing trace levels of hydrogen gas, which provides the energy to fix CO₂ via the Calvin–Benson–Bassham cycle, aiding their survival in nutrient-poor soils [77], such as those of the Terrace sites.

4.3. Exploring Major Metabolic Pathways of Prokaryotic Communities in MAG Agroecosystems

The results of our functional study on the prokaryotic communities of the Mixteca Alta Geopark reveal distinct metabolic capacities in the agroecosystems studied. Specifically, the identification of pathways associated with antibiotic biosynthesis (ansamycins, vancomycin, streptomycin) across the agroecosystems (Figure 6) highlights the role of soil microbial communities in producing secondary metabolites with antimicrobial properties. Moreover, research suggests that such antibiotic production is essential not only for microbial competition but also in plant–microbe interactions, influencing plant health and soil fertility [78]. Studies have demonstrated that soil bacteria, such as Actinobacteria, Proteobacteria, and Streptomyces, are involved in producing these metabolites, which are crucial for managing plant pathogens [79,80]. Thus, the presence of these pathways in MAG agroecosystems likely supports soil health and crop productivity by providing natural disease resistance mechanisms.

Furthermore, the biosynthesis of amino acids such as glutamine, glutamate, valine, leucine, and isoleucine in MAG soils indicates active nitrogen cycling and protein synthesis in the soil microbiome. For instance, microbial cells release amino acids through lysis or efflux, which are then broken down by extracellular enzymes like amino acid oxidase. This process produces ammonium nitrogen, which plants can readily absorb [81]. Additionally, amino acid biosynthesis has also been shown to play an important role in root colonization by bacteria [82], promoting plant growth by improving shoot and root development, and increasing water and mineral uptake [83].

In addition, pathways for pantothenate (vitamin B5) and lipoic acid metabolism are critical for energy production, coenzyme synthesis (like CoA), and non-enzymatic antioxidant functions [84,85,86]. Lipoic acid, a cofactor for pyruvate dehydrogenase and glycine decarboxylase, plays a vital role in energy metabolism. Similarly, it also acts as a non-enzymatic antioxidant, aiding plants under stress, such as drought, by mitigating adverse effects on photosynthesis. For example, in maize seedlings, lipoic acid was shown to reduce the impact on pigment (chlorophyll) content under drought conditions [87]. Pantothenate, a metabolite secreted by plant growth-promoting bacteria (PGPB), is a precursor for coenzyme A. Notably, its stimulative effect on plant growth has been observed in alfalfa seedlings, potatoes, and other green plants [88].

On the other hand, in addition to the general metabolic functions observed in the three MAG agroecosystems, we identified functional differences among them (Figure 7). These differences highlight that the agroecosystems (Lama-bordo, Terrace, and Valley) in the MAG have distinct dominant metabolic functions, suggesting that the soil microbiome is highly adapted to the specific environmental conditions of each site. In the case of Lama-bordo, the significant presence of lipopolysaccharide biosynthesis, lipoic acid metabolism, and fatty acid metabolism suggests that this ecosystem supports bacteria involved in soil formation, antioxidant functions, and plant disease control, potentially reflecting a nutrient-rich environment. Members of the families Gemmatimonadaceae and Sphingomonadaceae, which are abundant in Lama-bordo (Figure 5D–F), are involved in lipopolysaccharide production. These families have been proven to stabilize soil and increase aggregate strength through the adhesive properties of lipopolysaccharides, which are beneficial for soil structural properties by gluing soil particles together. The production of lipopolysaccharides requires high amounts of carbon, as found in nutrient-rich soils [88]. Fatty acids have been implicated in disease control in soils by suppressing soil-borne plant diseases [89], and there is growing interest in using fatty acid microbial groups for soil health assessments [90]. While lipoic acid functions as an antioxidant, helping plants under stress—such as drought—by mitigating the adverse effects on photosynthesis, as mentioned earlier. At the Valley site, the predominance of valine, leucine, and isoleucine biosynthesis, along with pantothenate and CoA biosynthesis, suggests an environment where microbial communities are focused on active nitrogen cycling and stimulating plant growth [81,85].

The dominance of pathways related to starch, sucrose, and galactose degradation, as well as bacterial chemotaxis in the Terrace area suggests a higher prevalence of bacteria involved in carbon cycling and microbial movement. Specifically, in the case of sugar degradation, the products released into the soil serve multiple key functions, including maintaining and stimulating microbial activity, which accelerates the decomposition of soil organic matter. This process releases stored nutrients such as nitrogen, phosphorus, and sulfur, while also providing a source of carbon and energy. Additionally, carbohydrates play a critical role in soil structure formation by binding mineral and organic particles, contributing to microaggregate formation, which is vital for soil development and stability [91]. In the case of bacterial chemotaxis, it is a mechanism associated with movement from low- to high-nutrient gradients. This allows bacteria to navigate non-homogeneous nutrient soils, such as those found in Terrace. Ecologically, this mechanism functions as a colonization strategy in microenvironments, helping bacteria escape stress factors, and play a significant role in biogeochemical cycles [92].

The metabolic profiles observed in Lama-bordo, Terrace, and Valley support the idea that specific agroecosystem management strategies in MAG directly influence microbial activity and soil health, exhibiting distinct metabolic functions tailored to their environmental conditions.

5. Conclusions

Distinct bacterial phyla, such as Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi, dominate the Mixteca Alta Geopark agroecosystems, supporting various ecological functions like organic matter decomposition, plant disease suppression, and nutrient cycling. By sustaining bacterial diversity, these agroecosystems ensure long-term soil health and resilience, emphasizing the need for sustainable management and conservation efforts to protect these ancient agricultural systems. The analysis of soil microbiomes reveals distinct phylogroups with a preferent association with the different agroecosystems of MAG, reflecting their unique environmental conditions. Nutrient-rich agroecosystems like Lama-bordo and Valley show a high abundance of copiotrophic bacteria such as Planctomycetes, Proteobacteria, and Gemmatimonadetes, which support soil health and nutrient cycling. In contrast, the Terrace site, with nutrient-poor soils, is dominated by oligotrophic Acidobacteria, essential for soil aggregation and adaptation to dry conditions. A diverse microbial community is often associated with resilient and productive soils, such as the Lama-bordos and Valley sites, which obtained the highest alpha diversity values and, at the same time, were the most similar in terms of their species composition and abundance, as observed in the DPCoA analysis. We also observed distinct metabolic capacities tailored to their environmental conditions. The microbial communities across these sites produce key secondary metabolites, including antibiotics that support plant health and natural disease resistance. Additionally, the metabolic pathways for amino acid biosynthesis, energy production, and carbohydrate degradation suggest active roles in nutrient cycling, soil structure formation, and plant growth promotion. The presence of WPS-2 (Eremiobacterota) and BRC1 phyla, which are poorly understood, indicates that there are still unknown aspects of the microbial communities in these agroecosystems, with the potential for undiscovered metabolic capabilities. Although their presence is minimal, it suggests the potential for unexplored microbial diversity in the GMA that may be adapted to specific environmental niches. The identification of both well-known and poorly understood microbial groups suggests that these agroecosystems are complex and potentially harbor novel microbial functions that could be important for sustainable agriculture and ecosystem conservation.

These findings highlight the significant influence of traditional land-use practices in shaping microbial diversity and activity, highlighting the ecological importance of prokaryotic diversity in maintaining soil fertility and agroecosystem resilience. By supporting bacterial diversity, these agroecosystems promote long-term soil health, underscoring the need for sustainable management and conservation efforts to protect these ancient agricultural systems. This is the first report on the agroecosystem microbiomes of MAG, which is characterized by its unique geodiversity and long history of human impact. Additionally, the study’s findings should be used to optimize management practices to enhance soil health, ecosystem resilience, and agricultural productivity in these ancient agricultural systems of the Mixteca Alta Geopark, as well as to highlight the importance of conserving microbial diversity as part of sustainable land management practices. Studying microbiomes also plays a crucial role in monitoring management practices, offering insights that can guide efforts to preserve and improve soil health while ultimately boosting agricultural productivity.