Diversity of Agaricomycetes at the forest-city interface of the Adolpho Ducke Forest Reserve, Manaus, Central Amazonia

Diversidade de Agaricomycetes em parcelas preservadas versus influenciadas pelo homem na Reserva Adolpho Ducke, Manaus (Amazônia Central)

Abstract

Macrofungal diversity is essential to forest ecosystems, supporting carbon cycling and plant community structure. However, anthropogenic disturbance threatens this diversity, particularly near urban areas. In Manaus, irregular settlements affect forest fragments such as the Adolpho Ducke Forest Reserve. This study described patterns of macrofungal diversity in preserved and anthropized sectors of an Amazonian forest. The study was conducted during a single field campaign in July 2024 and compared two sectors of the Adolpho Ducke Forest Reserve: one preserved forest sector and one anthropized sector. In each sector, 600 m² were surveyed across six 10 × 10 m plots. Sampling was performed through active searches along predefined transects, using standardized field effort per plot and the same field observer/team in both sectors. Fungal diversity was analyzed using species accumulation data, morphological and molecular identification, including phylogenetic tree construction and quantification of basidiomata per taxon, and ecological indices (Shannon-Wiener diversity index, Pielou’s evenness, and Sørensen similarity index). A total of 91 basidiomata and 31 taxa were recorded in the preserved sector, with Marasmius spp., Mycena spp., Amauroderma spp., Auricularia spp., and Schizophyllum spp. as the most frequent taxa. In the anthropized sector, 15 basidiomata and 8 taxa were recorded, with Trametes sp., Marasmius puttemasii, Schizophyllum sp., Pycnoporus sp., and an unidentified Agaricales morphotype as the most frequent taxa. Ecological index analysis indicated higher diversity in the preserved sector (H′ = 3.24) and slightly lower Pielou’s evenness (J) = 0.94, whereas the anthropized sector showed lower diversity (H′ = 1.97) and slightly higher Pielou’s evenness (J) = 0.95. These results suggest that anthropogenic influence may be associated with reduced macrofungal diversity at the forest-city interface.

Keywords:
anthropogenic impact; Agaricomycetes; Basidiomycota; macrofungi diversity

Resumo

A diversidade de macrofungos é essencial para os ecossistemas florestais, sustentando o ciclo do carbono e a estrutura das comunidades vegetais. No entanto, as influências antrópicas ameaçam essa diversidade, especialmente em áreas próximas a centros urbanos. Em Manaus, ocupações irregulares afetam fragmentos florestais, como a Reserva Florestal Adolpho Ducke. Este estudo descreveu padrões de diversidade de macrofungos em setores preservado e antropizado de uma área de floresta amazônica. O estudo foi conduzido em uma única campanha de campo, em julho de 2024, comparando dois setores da Reserva Florestal Adolpho Ducke: um setor de floresta preservada e um setor antropizado. Em cada setor, foram amostrados 600 m², distribuídos em seis parcelas de 10 × 10 m. A coleta foi realizada por busca ativa ao longo de transectos previamente definidos, com esforço amostral padronizado por parcela e pelo mesmo observador/equipe de campo em ambos os setores. A diversidade fúngica foi analisada por meio de curva de acumulação, identificação morfológica e molecular (incluindo construção de árvore filogenética e quantificação de basidiomas por táxon) e índices ecológicos (índice de diversidade de Shannon-Wiener, equitabilidade de Pielou e índice de similaridade de Sørensen). Como resultado, foram registrados 91 basidiomas e 31 táxons na área preservada, sendo Marasmius spp., Mycena spp., Amauroderma spp., Auricularia spp. e Schizophyllum spp. os táxons mais frequentes. Na área antropizada, foram registrados 15 basidiomas e 8 táxons, com destaque para Trametes sp., Marasmius puttemasii, Schizophyllum sp., Pycnoporus sp. e um morfótipo não identificado de Agaricales. A análise dos índices ecológicos indicou maior diversidade no setor preservado (H′ = 3.24) e menor uniformidade de Pielou (J) = 0.94, enquanto o setor antropizado apresentou menor diversidade (H′ = 1.97) e maior uniformidade de Pielou (J) = 0.95. Os resultados sugerem que a influência antrópica pode estar associada à menor diversidade de macrofungos no setor antropizado da interface floresta-cidade.

Palavras-chave:
impacto antrópico; Agaricomycetes; Basidiomycota; diversidade de macrofungos

1. Introduction

Macrofungi, particularly those belonging to Basidiomycota, play a central role in forest ecosystems by driving organic matter decomposition, nutrient cycling, and plant community dynamics. These organisms are essential for maintaining ecosystem functioning, especially in tropical forests, where biodiversity and biological interactions are highly complex. However, anthropogenic pressures, including urban expansion, deforestation, and land-use change, have increasingly been associated with changes in fungal diversity and community structure. In tropical regions such as the Amazon, these impacts may be intensified by rapid and often unplanned urban growth, particularly in areas located at the interface between preserved forests and urban environments.

Previous studies have shown that anthropogenic disturbance and urbanization can alter fungal and macromycete communities, affecting richness, functional diversity, and community composition in tropical and subtropical ecosystems, including Amazonian forests (Abrego et al., 2020; Gómez-Hernández et al., 2021; López-Quintero et al., 2012). In macrofungal communities, habitat transformation may reduce species richness, modify functional composition, and favor taxa associated with disturbed substrates or simplified habitats (Gómez-Hernández et al., 2021; López-Quintero et al., 2012; Mueller et al., 2016).

In Manaus, the irregular occupation of Environmental Protection Areas (EPAs), lack of basic infrastructure, and limited availability of essential services are consequences of inadequate urban planning (Pires Gaspar et al., 2019). These factors directly affect local ecosystems, where the uncontrolled growth of informal settlements and slums increases pressure on fungal biodiversity in preserved areas (Gomes et al., 2022). The Adolpho Ducke Forest Reserve (RFAD), which covers 100 km² and is located in the peripheral neighborhoods of northern Manaus (Costa et al., 2019), has experienced degradation due to human activity, particularly between 1984 and 2023 (Souza et al., 2023). Ongoing encroachment exacerbates deforestation and pollution, subjecting the area to intense anthropogenic pressure that may affect fungal communities. However, this impact remains insufficiently investigated.

For consistency with Brazilian environmental law, the term environmental degradation is used here in accordance with Law No. 6,938/1981 (Brasil, 1981), Article 3, which defines pollution as the degradation of environmental quality resulting from activities that directly or indirectly impair human health, safety, and welfare; create adverse conditions for social and economic activities; affect biota; affect the aesthetic or sanitary conditions of the environment; or release matter or energy in disagreement with established environmental standards. In this study, anthropogenic influence refers to the local effects observed at the forest-city interface, including vegetation removal, edge exposure, soil exposure, waste deposition, altered microclimate, and disturbance of decomposing substrates.

Previous field-based documentation from the Adolpho Ducke Forest Reserve has reported high diversity of macroscopic fungi in the area; however, comparative studies focusing on macrofungal assemblages across preserved and anthropized sectors at the forest-city interface remain scarce (Braga-Neto et al., 2008).

Thus, forest degradation on the outskirts of RFAD may be contributing to the disappearance of several macrofungal species that are essential for regulating the regional ecosystem and that may have significant potential for future applications in industry and healthcare. Understanding how human activities affect fungal diversity in this region is crucial for investigating potential biodiversity changes in response to global anthropogenic alterations. In this context, diversity studies conducted in environmental protection areas, such as RFAD, play a fundamental role in assessing the impacts of degradation, particularly in tropical regions such as the Amazon Rainforest. This study therefore aimed to contribute to the understanding of anthropogenic and pollution-related impacts on regional macrofungal diversity. Specifically, we explored whether the anthropized sector showed lower macrofungal diversity and altered community composition compared with the preserved sector.

2. Materials and Methods

2.1. Study location and sampling design

The Adolpho Ducke Forest Reserve (RFAD) is a ~100 km² (10,000 ha) protected terra firme rainforest on the northeastern edge of Manaus (Amazonas, Brazil), administered by INPA/MCTI. The reserve is widely used as a long-term ecological research site and comprises old lateritic uplands dissected by small streams (igarapés), with closed-canopy ombrophilous forest and a humid equatorial climate.

The study was conducted in July 2024, during the relatively drier period in Manaus. We surveyed two contrasting sectors within RFAD: a preserved forest sector (−2.967895° S, −59.931167° W) and a sector under marked anthropogenic influence (−2.990801° S, −59.970361° W). In each sector, sampling covered 600 m² distributed across six permanent 10 × 10 m plots (100 m² each). Figure 1 shows the location of the study area in RFAD within the Manaus urban matrix. This descriptive and exploratory design therefore represents a comparison between two sectors of RFAD rather than a replicated comparison among multiple independent forest areas. The preserved and anthropized sectors were treated as the main comparative units, while the six plots within each sector were used as subsampling units. Consequently, no inferential statistical test was applied to claim significance between conditions.

Figure 1
Geospatial context of the Adolpho Ducke Forest Reserve (Manaus, Brazil): delineation of preserved and anthropized study sectors and sampling extents. Coordinates are given in decimal degrees (latitude, longitude). Preserved sector — Plot 1: −2.967895° S, −59.931167° W; Plot 2: −2.967964° S, −59.930987° W; Plot 3: −2.968009° S, −59.930695° W; Plot 4: −2.967184° S, −59.931028° W; Plot 5: −2.968369° S, −59.930858° W; Plot 6: −2.967771° S, −59.931568° W. Anthropized sector — Plot 1: −2.990801° S, −59.970361° W; Plot 2: −2.991270° S, −59.970164° W; Plot 3: −2.991173° S, −59.969953° W; Plot 4: −2.990754° S, −59.970000° W; Plot 5: −2.990755° S, −59.970724° W; Plot 6: −2.991577° S, −59.970247° W.

In this manuscript, the sector under marked anthropogenic influence is hereafter referred to as the anthropized sector, whereas the forest sector with lower visible disturbance is referred to as the preserved sector.

We assessed vegetation condition using two standard spectral indices derived from satellite reflectance. NDVI (Normalized Difference Vegetation Index) compares near-infrared (NIR), which is strongly reflected by healthy leaves, with red light, which is strongly absorbed by chlorophyll, to indicate greenness and photosynthetic vigor. EVI (Enhanced Vegetation Index) also uses blue reflectance to better correct for atmospheric effects and soil/background influence, improving sensitivity in high-biomass forests. We used Sentinel-2 Level-2A imagery (10 m) and processed it in Google Earth Engine (Gorelick et al., 2017). Scenes were filtered by date and area, clouds and cirrus were masked using QA60, and a median mosaic was created. The indices were then computed from surface reflectance bands B8 (NIR), B4 (red), and B2 (blue) using the standard Formulas 1 and 2:

$\text{NDVI}=(\text{NIR}-\text{Red})/(\text{NIR}+\text{Red})$ (1)

and

$$\begin{array}{c}EVI=2.5\times \left(NIR-Red\right)/\\ \left(NIR+6\times Red-7.5\times Blue+1\right)\end{array}$$ (2)

Figure 2 presents vegetation conditions analyzed from satellite imagery using Google Earth Engine.

Figure 2
Vegetation condition based on satellite imagery acquired shortly after the July 2024 fungal sampling campaign (Normalized Difference Vegetation Index [NDVI] and Enhanced Vegetation Index [EVI]; 2-28 August 2024), together with field measurements of relative humidity, air temperature, and soil pH, contrasting the anthropized (A) and preserved (B) sectors of the Adolpho Ducke Forest Reserve.

Anthropized sector: This sector is located on the outskirts of RFAD near “Campo do Flamengo” (Santa Etelvina, Manaus), with an open physiognomy dominated by grasses/shrubs and scattered trees (canopy ≤ 12 m; e.g., Mangifera spp., Anacardium spp., and Malpighia spp.). Litter is sparse, and the soil is more exposed. Sentinel-2 imagery (COPERNICUS/S2_SR_HARMONIZED) filtered in Google Earth Engine indicated NDVI = 0.15-0.40 and EVI ≤ 0.40; field microclimate during surveys was RH = 56-62% and T = 30-34 °C; soil pH = 3.54.

Preserved sector: This sector is located ~5 km inside RFAD and is characterized by dense ombrophilous forest with a canopy of ~32 m. A thick leaf-litter layer, notably from Aldina spp. and Astrocaryum spp., and small watercourses occur along the transects. Sentinel-2 products from the same 2024 window showed NDVI = 0.58-1.00 and EVI up to 1.00; field microclimate was RH = 74-87% and T = 26-28 °C; soil pH = 4.00.

2.2. Fungal collection

Procedures followed previous work. During the single July 2024 field campaign, basidiomata were sampled by active search in each 10 × 10 m plot. In each plot, the field observer/team followed predefined transects and visually inspected leaf litter, soil, decaying wood, fallen trunks, and other available substrates using the same standardized search effort in both sectors. Upon arrival at each plot, the observer walked the predefined transect and grouped all basidiomata into morphotypes using consistent macromorphological traits, including pileus shape, size, and texture; stipe features; hymenophore type and color; odor or latex when present; and substrate.

For each morphotype, we counted the number of independent basidiomata recorded during the same visit. These counts represent field abundance/occurrence and provide a faithful description of the community as encountered in situ.

For downstream laboratory work, we collected up to five basidiomata per morphotype, when available, and recorded GPS coordinates, microhabitat, and substrate. In the laboratory, one to five representatives per morphotype were prioritized for DNA extraction and ITS amplification. Because the INPA Laboratory of Mycology collection preserves cultivable fungi, living cultures were obtained and maintained for the cultivable isolates recovered in this study. Among the 33 morphotypes recorded in the field, 14 cultivable isolates yielded successful ITS-PCR amplification and sequencing. These isolates were provisionally preserved in the Laboratory of Mycology collection under LM codes within the interval LM6227-LM6241. Field records, morphological characterization, ITS sequences, GenBank accession numbers, and molecular identification data are provided in the manuscript, Supplementary File S1 and Supplementary Table S2, and Supplementary Table S3, ensuring reproducibility of the taxonomic assignments.

2.3. Fungal identification

2.3.1. Morphological identification

Morphological identification followed Lodge et al. (2004). Initial in situ identification was based on macromorphological characteristics, including pileus diameter, shape, color, surface, texture, and margin; hymenium shape, color, spacing, and attachment; and stipe length, diameter, shape, color, texture, and the presence of a ring or volva. Micromorphological identification, carried out at the Mycology Laboratory (INPA), considered spores, basidia, including number and shape of sterigmata, cystidia, and hyphal structure. Microstructures were visualized using an Axioskop 40 microscope (Zeiss, Oberkochen, Germany). Lactophenol cotton blue stain and a 3.0% potassium hydroxide (KOH) solution were used to facilitate visualization of microstructures.

Morphological identification was also supported by taxonomic keys and reference descriptions for macroscopic Basidiomycota, including field and microscopic characters used for agaricoid, polyporoid, auricularioid, and other Agaricomycetes groups. The characters compared included pileus morphology, hymenophore type, lamellae or pore configuration, stipe features, basidiome consistency, substrate, spore morphology, basidia, cystidia, and hyphal structures. The taxonomic keys and reference sources used to support each morphological assignment are detailed in Supplementary File S1.

2.3.2. Molecular identification

For DNA extraction, approximately 80 mg of biomass from the pileus and stipe of each macrofungal sample was subjected to the phenol-chloroform-isoamyl alcohol extraction method (Ferrer et al., 2001). For conventional polymerase chain reaction (PCR), we followed White et al. (1990). A final reaction volume of 30 μL was prepared for each sample, containing 2.0 mM MgCl2, 200 μM dNTPs (ThermoFisher Scientific, Massachusetts, USA), 1.5 U/μL Platinum II Taq polymerase enzyme (ThermoFisher Scientific, Massachusetts, USA), and 0.5 μM each of primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). PCR was performed using a Veriti 96 thermal cycler (Applied Biosystems, Foster City, USA), programmed with an initial step at 94 °C for 10 min, followed by 35 cycles of 94 °C for 1 min, 56 °C for 1 min 30 s, and 72 °C for 1 min. A final extension was then performed at 72 °C for 10 min.

For electrophoresis, a 1.5% agarose gel was prepared in 50 mL of 1× TBE buffer obtained by diluting 10× TBE (Invitrogen, California, USA) 1:10 with distilled water. For sample loading, 2 μL of 6× DNA Gel Loading Dye (ThermoFisher Scientific, Massachusetts, USA) was added to 8 μL of amplicon, and 8 μL of Invitrogen SYBR Safe DNA Gel Stain (ThermoFisher Scientific, Massachusetts, USA) was used in the gel. Electrophoresis was performed at 100 V and 100 mA for 40 min. A 100 bp ladder (ThermoFisher Scientific, Massachusetts, USA) was used in all runs.

The amplified products were purified using a polyethylene glycol (PEG) solution (20% PEG and 2.3 M NaCl) mixed with the sample at a 1:1 ratio, followed by incubation at 37 °C for 15 min in a thermal block. The samples were then centrifuged at 6,000 rpm for 15 min at 15 °C. The supernatant was discarded, and 125 μL of 80% ethanol was added. The samples were centrifuged again at 6,000 rpm for 15 min at 15 °C. The supernatant was removed, and the sample was incubated at 60 °C for 15 min. Finally, 30 μL of Tris-HCl 10 mM and EDTA 1 mM solution were added for elution.

Sequencing was conducted in collaboration with the Molecular Biology Thematic Laboratory (LTBM) at INPA using an ABI Prism 377 sequencer (Applied Biosystems, Foster City, USA). The resulting sequences were analyzed using BioEdit 7.7.1 (Hall, 1999) and compared with sequences in the NCBI database using BLAST (Basic Local Alignment Search Tool) for taxonomic identification. The sequences with the highest similarity were considered for identification.

2.3.3. Phylogenetic analysis

The ITS sequences obtained in this study were compared with reference sequences retrieved from GenBank based on BLASTn similarity and taxonomic relevance. Multiple sequence alignment was performed using the MUSCLE algorithm (Edgar, 2004), followed by manual inspection and trimming of ambiguous regions. Phylogenetic reconstruction was conducted in MEGA X using the Maximum Likelihood (ML) method, with the Tamura-Nei model including gamma-distributed rates and invariant sites (G+I), and bootstrap support was estimated from 1,000 replicates (Kumar et al., 2018). The ML tree was used as an ITS-based molecular identification framework for taxonomic placement of the isolates. To avoid using a distant Ascomycota outgroup, the tree was rooted with non-Agaricomycetes Basidiomycota reference taxa positioned outside the focal Agaricomycetes clade, including representatives of Wallemiomycetes, Geminibasidiomycetes, Dacrymycetes, Tremellomycetes, and Bartheletiomycetes, as shown in Figure 3. Branches with bootstrap support below 50% were collapsed in the consensus tree (Kumar et al., 2018). Species-level identifications were accepted only when ITS similarity, phylogenetic placement, and morphological evidence were concordant; when this evidence was insufficient, taxa were conservatively treated at the genus level or as morphotypes. The analysis was interpreted as a taxonomic identification framework rather than as a deep phylogenetic reconstruction among Basidiomycota classes.

Figure 3
ITS-based Maximum Likelihood phylogenetic tree showing the placement of Agaricomycetes isolates obtained from preserved and anthropized plots at the Adolpho Ducke Forest Reserve, Manaus, Central Amazonia. Reference sequences retrieved from GenBank are indicated by accession numbers. Red squares indicate isolates from preserved-sector samples, and brown squares indicate isolates from anthropized-sector samples. Non-Agaricomycetes Basidiomycota reference taxa representing Wallemiomycetes, Geminibasidiomycetes, Dacrymycetes, Tremellomycetes, and Bartheletiomycetes were included as external reference taxa/outgroups to root the tree. Bootstrap values are shown at nodes when ≥50%. The tree is intended to support molecular taxonomic assignment based on ITS sequences and should not be interpreted as a robust reconstruction of deep relationships among Basidiomycota classes.

2.4. Analysis of diversity indices

All basidiomata were counted per plot and grouped by morphotype to enable descriptive quantitative (abundance) and qualitative (trait) analyses. Each basidioma was treated as one counting unit; clusters of pilei were counted as separate basidiomata, whereas contiguous resupinate patches were counted as one record per substrate. Under our experimental conditions, “taxon” or “morphotype” refers to the lowest rank resolved by morphology and/or ITS sequencing (López-Quintero et al., 2012; Farias et al., 2025). Sector-level richness and abundance data were summarized in Supplementary Table S3 to increase transparency, but plot-level values were not treated as independent replicates for inferential testing.

Fungal diversity was quantified using the Shannon-Wiener diversity index (H′), Pielou’s evenness (J), and Sørensen similarity index (S). Indices were calculated in Microsoft Excel (Microsoft Corporation, Redmond, USA).

Shannon diversity (H′): We computed H′ = −Σ(pᵢ × ln pᵢ), where pᵢ = nᵢ/N, nᵢ is the number of basidiomata of the i-th taxon, and N is the total number of basidiomata in the sample. Higher H′ indicates greater heterogeneity and diversity. Interpretation: low H′ indicates dominance by few taxa (low richness and/or strong unevenness), whereas high H′ reflects many taxa with more balanced abundances. Communities with similar richness can still differ in H′ if abundances are more even in one of them.

Evenness (Pielou’s J): To assess the evenness of abundances among taxa, we used J = H′/ln S, where S is taxon richness (number of taxa in the sample). J ranges from 0 (strong dominance) to 1 (equal abundances). Interpretation: values near 0 indicate that a few taxa concentrate most basidiomata, whereas values near 1 indicate a more uniform distribution, allowing direct comparison of evenness regardless of differences in richness.

Sørensen similarity (S): Community similarity between the preserved and anthropized sectors was estimated using the Sørensen-Dice coefficient: S = 2c/(a + b), where a and b are the numbers of taxa observed in communities 1 and 2, respectively, and c is the number of taxa shared by both. S ranges from 0 (no shared taxa) to 1 (identical sets of taxa). Interpretation: values < 0.3 suggest marked turnover in taxon composition; intermediate values (~0.3-0.6) indicate partial overlap; and values > 0.6 denote high similarity in species lists between communities.

2.5. Sampling unit and analytical scope

The sampling units in this study consisted of two 600 m² sampled sectors, one preserved sector and one anthropized sector, within the Adolpho Ducke Forest Reserve. Although each sector included six 10 × 10 m plots, these plots were treated as subsampling units within each sector rather than as independent replicated forest areas. Therefore, no inferential statistical test was used to claim significant differences between conditions; all diversity indices and plot-level data are presented as descriptive patterns. This analytical scope was adopted because the study was based on one preserved sector and one anthropized sector sampled during a single field campaign.

Sector-level richness and abundance were retained as descriptive transparency data in Supplementary Table S3, and ecological indices were interpreted at the sector level.

The phylogenetic component is described in Section 2.3.3 and was used only to support ITS-based taxonomic placement of selected isolates, not to test diversity differences among sectors.

3. Results

3.1. Fungal collection

To investigate the influence of anthropogenic activity on Agaricomycetes diversity, fungi were collected and identified across an area of 1,200 m². Of this total, 600 m² were characterized as having low anthropogenic influence, while 600 m² were classified as having high anthropogenic influence. In the preserved sector, 91 basidiomata and 31 taxa were recorded, whereas only 15 basidiomata and 8 taxa were identified in the anthropized sector.

3.2. Fungal identification

The taxa observed in this study varied between the preserved and anthropized sectors. In the preserved sector, Mycenaceae and Marasmiaceae predominated, whereas Polyporaceae was more prevalent in the anthropized sector (Table 1) (Figure 4). Identification records containing macromorphological and micromorphological data for each taxon are available in Supplementary File S1. Of the 33 morphotypes described in the present study, samples from 14 were viable for sequencing analysis. These 14 cultivable isolates were maintained as living cultures and provisionally preserved in the Laboratory of Mycology collection under LM codes within the interval LM6227-LM6241. Molecular identification supported taxonomic assignments at the species or genus level depending on the concordance among ITS data, phylogenetic placement, and morphology (Supplementary Table S2). Taxa without sufficient evidence for species-level resolution were retained at the genus level or as morphotypes, ensuring correspondence among Table 1, Supplementary File S1, and Supplementary Table S2.

Table 1
Absolute counts (number of basidiomata) and relative frequencies (%) of macrofungal taxa by sector (preserved vs. anthropized), pooled across six 10 × 10 m plots per sector; total basidiomata N = 91 (preserved) and N = 15 (anthropized).

Taxa	Preserved sector		Anthropized sector
	FAB	Freq (%)	FAB	Freq (%)
Marasmius sp. (2)	6	6.59	0	0
Marasmius cf. haematocephalus	6	6.59	0	0
Mycena sp. (1)	6	6.59	0	0
Mycena sp. (2)	6	6.59	0	0
Marasmius cf. puttemasii	6	6.59	2	13.33
Marasmiellus praecutus	5	5.49	0	0
Mycena sp. (3)	5	5.49	0	0
Schizophyllum sp. (1)	5	5.49	2	13.33
Fomitopsis modesta	5	5.49	1	6.67
Marasmius sp. (1)	4	4.40	0	0
Amauroderma sp. (1)	3	3.30	0	0
Auricularia sp. (1)	3	3.30	0	0
Marasmius cf. bellus	3	3.30	0	0
Tetrapyrgos sp.	3	3.30	0	0
Amauroderma sp. (2)	2	2.20	0	0
Auricularia sp. (2)	2	2.20	0	0
Favolus sp.	2	2.20	0	0
Hexagonia sp.	2	2.20	0	0
Marasmius rotula	2	2.20	0	0
Pycnoporus sp.	2	2.20	2	13.33
Trametes sp.	2	2.20	4	26.67
Agaricales morphotype (unidentified)	2	2.20	2	13.33
Amauroderma sp. (3)	1	1.10	0	0
Gymnopus talisiae	1	1.10	0	0
Geastrum hirsutum	1	1.10	0	0
Hygrocybe sp. (1)	1	1.10	0	0
Lentinus sp.	1	1.10	0	0
Cystolepiota sp.	1	1.10	0	0
Leucoagaricus leucothites	1	1.10	0	0
Leucoagaricus sp.	1	1.10	0	0
Pleurotus sp.	1	1.10	0	0
Hygroaster cf. albellus	0	0	1	6.67
Schizophyllum sp. (2)	0	0	1	6.67
TOTAL	91	100	15	100

• Note: Order-level identifications were retained when genus-level resolution was not possible. FAB = frequency of basidiomata, expressed as the absolute number of basidiomata recorded per taxon. Taxonomic assignments follow the conservative nomenclature adopted in Supplementary Files S1 and S2; “cf.” indicates taxa with morphological and/or ITS evidence close to the listed species but without unequivocal species-level confirmation.

Figure 4
Representative basidiomata of the most prevalent macrofungal taxa recorded in preserved and anthropized sectors of the Adolpho Ducke Forest Reserve, Manaus, Central Amazonia. (A) Mycena sp. (1); (B) Mycena sp. (2); (C) Marasmius sp. (2); (D) Marasmius puttemasii; (E) Trametes sp.; and (F) Pycnoporus sp. These taxa were selected based on their relative abundance in the preserved and/or anthropized sectors.

Figure 3 presents an ITS-based Maximum Likelihood phylogenetic tree showing the molecular placement of the Agaricomycetes isolates analyzed in this study. Reference sequences retrieved from GenBank were included to support species- or genus-level taxonomic assignment, and non-Agaricomycetes Basidiomycota reference taxa were included as external reference taxa/outgroups to root the tree. The tree was interpreted as a molecular identification framework and not as a robust reconstruction of deep evolutionary relationships among Basidiomycota classes.

3.3. Species accumulation and plot-level descriptive data

The taxon-accumulation curves are presented in Supplementary Figure S3, while the corresponding sector-level summary data are provided in Supplementary Table S3. Because this study was based on a single preserved sector and a single anthropized sector, with plots treated as subsampling units, plot-level richness and abundance were used only as descriptive information and not as independent replicates for inferential testing. Given the low number of basidiomata in the anthropized sector, plot-level values are presented only for transparency and interpreted cautiously.

3.4. Diversity indices

To assess this variation, Agaricomycetes diversity was described using the Shannon-Wiener diversity index (H′), Pielou’s evenness (J), and Sørensen similarity index (S) (Table 2). In addition, the five most abundant taxa in each sector were identified to determine dominant taxa within distinct ecological contexts. These analyses were used to describe how anthropogenic disturbance was associated with fungal diversity and community structure.

Table 2
Alpha diversity (Shannon-Wiener H′, natural log), Pielou’s evenness (J), and between-sector Sørensen similarity (S) for macrofungal communities in the preserved and anthropized sectors of the Adolpho Ducke Forest Reserve, computed from basidiomata counts.

Parameter	Preserved sector	Anthropized sector
Sector-exclusive taxa	25	2
Taxa shared by both sectors	6
Five most abundant taxa	Mycena sp. (1)	Trametes sp.
	Mycena sp. (2)	Marasmius cf. puttemasii
	Marasmius sp. (2)	Pycnoporus sp.
	Marasmius cf. puttemasii	Schizophyllum sp.
	Marasmius cf. haematocephalus	Agaricales morphotype (unidentified)
Shannon-Wiener (H′)	3.24	1.97
Pielou’s evenness (J)	0.94	0.95
Sørensen similarity (S)	0.308

The preserved sector exhibited higher fungal diversity (H′ = 3.24) than the anthropized sector (H′ = 1.97), indicating a descriptive pattern of reduced diversity in the anthropized sector. Evenness values were similar between sectors, with J = 0.94 in the preserved sector and J = 0.95 in the anthropized sector. Sørensen’s similarity index (S = 0.308) indicated limited overlap between these environments, emphasizing the role of habitat integrity in maintaining fungal diversity (Table 2).

4. Discussion

This study revealed differences in macrofungal diversity between the preserved and anthropized sectors. Mycenaceae and Marasmiaceae predominated in the preserved sector, whereas Polyporaceae was more frequent in the anthropized sector. The combined morphological and molecular analyses supported the taxonomic identification of macrofungi and were consistent with the higher richness observed in the preserved sector. Among the 33 morphotypes recorded, 14 were viable for sequencing. Most sequenced taxa were obtained from the preserved sector, whereas the anthropized sector included selected taxa such as Fomitopsis modesta. Therefore, the molecular dataset should be interpreted as taxonomic support for selected morphotypes rather than as a complete representation of sector-exclusive diversity. Sequencing and morphological analyses resulted in the identification of eight species and six genera. These findings underscore the importance of environmental conservation and suggest that anthropogenic disturbance may be associated with changes in macrofungal biodiversity.

Under the experimental conditions, the preserved sector exhibited a higher number of basidiomata and taxa. Samples were collected from a 1,200 m² area, with 600 m² in the preserved sector yielding 91 basidiomata and 31 taxa, whereas the anthropized sector yielded 15 basidiomata and 8 taxa. Sector-level summary data provided in Supplementary Table S3 indicated a greater cumulative number of taxa in the preserved sector. Similar patterns have been reported in studies comparing urbanized or anthropized environments with less disturbed tropical and subtropical areas (Abrego et al., 2020; Gómez-Hernández et al., 2021; Yusran et al., 2022). Understanding anthropogenic impacts on tropical ecosystems through the integration of morphological and molecular data is crucial for conservation, environmental monitoring, mitigation of edge degradation, recovery actions, and exploration of the biotechnological potential of fungal species.

In the preserved sector, the families Mycenaceae and Marasmiaceae predominated, whereas Polyporaceae was more prevalent in the anthropized sector. Taxonomic composition analysis revealed that Marasmius spp. was the most abundant genus in the preserved sector, representing 29.67% of the recorded basidiomata. In the anthropized sector, Trametes sp. was the most abundant macrofungal taxon, accounting for 26.67% of the recorded basidiomata. Our findings are consistent with a substrate-driven pattern: leaf-litter decomposers prevailed in the preserved sector, while in the anthropized sector the loss of canopy inputs may have reduced litter availability, and standing or fallen trunks may have become the principal substrate, favoring wood-decaying genera. This interpretation is also consistent with studies on wood-decay fungal communities, which show that substrate availability, habitat condition, and disturbance gradients can influence the distribution and composition of lignicolous Agaricomycetes and other decomposer fungi (Dossa et al., 2021; Farias et al., 2025). Rodrigues et al. (2023) identified a decline in macrofungal richness in disturbed areas of South American biomes, emphasizing the loss of species dependent on preserved microhabitats.

The ITS molecular marker enabled the distinction of the macrofungi studied here at the genus level and, for some taxa, at the species level within Agaricomycetes. However, molecular identification based on a single marker has limitations, especially in groups with low ITS divergence, incomplete reference databases, or unresolved species complexes. Therefore, ITS-based assignments were interpreted together with morphological evidence, and species-level names were retained only when molecular and morphological data were concordant. Taxa that could not be confidently resolved at the species level were treated conservatively at the genus level or as morphotypes. These findings are consistent with studies conducted in China by He and Zhao (2022) and Xu et al. (2025), which investigated macrofungal taxonomic diversity using the ITS region as a molecular marker. Previous studies highlight the need for additional markers to achieve more precise species-level identification within this group and to better understand evolutionary relationships among genera and families (Tedersoo et al., 2018).

The difference in Shannon-Wiener index values (3.24 in the preserved sector and 1.97 in the anthropized sector) suggests that human influence may be associated with lower macrofungal diversity. This pattern agrees with previous studies showing that disturbance, land-use change, and urbanization can reduce macrofungal richness and alter community composition (López-Quintero et al., 2012; Gómez-Hernández et al., 2021; Yusran et al., 2022). The Sørensen similarity index (S = 0.308) indicated low taxonomic similarity between sectors, particularly for the genera Marasmius spp. and Mycena spp., which are associated with leaf litter in tropical and subtropical ecosystems. The predominance of wood-decaying fungi in the anthropized sector may reflect the increased availability of cut or fallen trunks associated with local disturbance.

The taxa recorded in this study may also have biotechnological relevance, although this potential was not experimentally evaluated here. Wood-decaying fungi such as Trametes, Pycnoporus, Fomitopsis, Schizophyllum, and related Polyporaceae are frequently associated with lignocellulose degradation and may be useful targets for future studies on oxidative enzymes, biodegradation, biomass transformation, and bioprocesses. Litter-associated genera such as Marasmius and Mycena may also contribute to organic matter turnover and represent promising sources for bioprospecting enzymes and secondary metabolites. These potential applications should be investigated in future culture-based, biochemical, and genomic studies before any applied claims are made.

Recent studies published in the Brazilian Journal of Biology also support the ecological and biotechnological relevance of fungi associated with Brazilian ecosystems, including fungi from Amazonian and Cerrado aquatic systems and decaying wood in the Brazilian Amazon (Souza et al., 2022; Canto et al., 2023).

Among the limitations of the present study, the survey was spatially and temporally restricted: we sampled two sectors within RFAD, totaling 600 m² per condition, during a single field window in July 2024. Consequently, our inferences may not capture heterogeneity at larger spatial scales across the reserve’s environmental gradients or seasonal turnover in macrofungal fruiting between wet and dry periods. Future work should expand plot networks beyond the current sectors and implement repeated surveys across seasons to resolve phenology and interannual variability. Molecularly, identification relied primarily on ITS rDNA and yielded sequences for a subset of morphotypes, which constrains species-level resolution in several groups and may bias diversity estimates. Adopting multilocus barcodes (e.g., LSU, RPB1/2, TEF1) and improving tissue preservation and extraction protocols could increase recovery and taxonomic precision. Finally, our design treated plots as subsamples within each sector; incorporating true replication at the sector level and complementary environmental covariates, such as microclimate and substrate inventories, would strengthen future assessments of the association between anthropogenic disturbance and macrofungal diversity.

Nonetheless, the study emphasizes the importance of environmental preservation for maintaining fungal biodiversity and reveals clear descriptive differences between preserved and anthropized sectors. These patterns reflect substantial environmental differences, such as higher temperature and lower humidity in the anthropized sector, which may influence the composition and functionality of fungal communities. Sequencing data supported the presence of species described in the literature and indicated the potential for identifying cryptic species, reinforcing the relevance of an integrated approach that combines molecular and morphological analyses.

5. Conclusion

This study showed that the preserved sector had greater diversity of Agaricomycetes macrofungi, with particular emphasis on Marasmius sp. and Mycena sp. Identification was achieved through morphological analysis and ITS sequencing. Ecological indices and species accumulation data indicated higher richness in the preserved sector, underscoring the importance of conservation for fungal biodiversity and future research in ecology and biotechnology. In contrast, the anthropized sector showed lower fungal occurrence, with reduced richness and abundance, a pattern consistent with the influence of human activity on fungal communities.

These findings reinforce the need for integrated conservation, monitoring, and environmental recovery actions to preserve fungal diversity and ecological balance. From a management perspective, the forest-city interface of RFAD should be prioritized for actions aimed at reducing edge degradation, vegetation removal, waste deposition, and disturbance of decomposing substrates. Long-term monitoring with repeated sampling across wet and dry seasons is also needed to evaluate temporal variation in macrofungal fruiting and strengthen ecological interpretations of anthropogenic influence.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary File S1

Identification sheets.

Supplementary Table S2

Study sequence GenBank accession numbers, conservative taxonomic assignments, closest ITS barcode matches, closest-match accession numbers, and sequence similarity values for 14 sequenced macrofungal isolates from the Adolpho Ducke Forest Reserve, Manaus, Amazonas, Brazil.

Supplementary Table S3

Sector-level richness and abundance recorded in the preserved and anthropized sectors of the Adolpho Ducke Forest Reserve, Manaus, Amazonas, Brazil.

Supplementary Figure S3

Taxon-accumulation curves for macrofungal records from the preserved and anthropized sectors of the Adolpho Ducke Forest Reserve, Manaus, Amazonas, Brazil.

This material is available as part of the online article from https://doi.org/10.1590/1519-6984.301973

Acknowledgements

J.F.V.E. was a scholarship recipient from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (Process No. 88887.821748/2023-00). This study was funded by the project “Production of Antimicrobials from Fungi Isolated from Water Samples of a Polluted Stream in Manaus, Amazonas,” coordinated by Dr. João Vicente Braga de Souza from the Laboratório de Micologia Médica, Instituto Nacional de Pesquisas da Amazônia (INPA), with support from the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) through EDITAL N. 010/2021 – CT&I PRIORITY AREAS. We thank FAPEAM for financial support, CAPES for the scholarship, INPA, and the Programa de Pós-Graduação em Genética, Conservação e Biologia Evolutiva (GCBEv) for their support and assistance. This study was funded by the project “Production of Antimicrobials from Fungi Isolated from Water Samples of a Polluted Stream in Manaus, Amazonas,” coordinated by Dr. João Vicente Braga de Souza from the Medical Mycology Laboratory at the National Institute for Amazonian Research (INPA), with support from the Amazonas State Research Support Foundation (FAPEAM) through EDITAL N. 010/2021 – CT&I PRIORITY AREAS.

Data Availability Statement

The datasetas generated and analyzed during the current study are available within the article and supplementary material. DNA sequences obtained from the fungal isolates have been deposited in the NCBI GenBank database under accession numbers PQ850553-PQ850566. These accession numbers correspond to 14 representative sequenced isolates obtained from preserved and anthropized sectors of the Adolpho Ducke Forest Reserve (Manaus, Central Amazonia, Brazil), as detailed individually in Supplementary Table S2. Table S2 provides detailed information for each isolate, including study sequence accession number, conservative taxonomic assignment, closest ITS match, closest-match accession number, and sequence similarity value. Sector-level richness and abundance data are provided in Supplementary Table S3, and the corresponding curves are shown in Supplementary Figure S3. The 14 cultivable isolates that yielded successful ITS-PCR amplification and sequencing were maintained as living cultures and provisionally preserved in the Laboratory of Mycology collection under LM codes within the interval LM6227-LM6241. Because these culture codes are preliminary and not yet individually linked to final curatorial accession numbers, they are reported here as a provisional deposit interval rather than as an additional supplementary table. Field records, morphological descriptions, ITS sequences, GenBank accession numbers, and sequence similarity values provide the basis for reproducibility of the taxonomic identifications presented in this study. All sequences are publicly accessible in GenBank, ensuring transparency and reproducibility of the molecular identifications presented in this study.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  17 Oct 2025
• Accepted
  01 June 2026