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. 2024 Feb 16;14(3):79. doi: 10.1007/s13205-024-03932-9

Diversity of soil fungi from sacred groves of Kerala, India revealed by comparative metagenomics analysis using illumina sequencing

Keerthana Nandakumar 1,✉, P V Anto 1, Ignatius Antony 1
PMCID: PMC10873253  PMID: 38371901

Abstract

The diversity, composition, and abundance of soil fungi from three sacred groves in Kerala, namely Iringole kavu of Ernakulam District, Kollakal Thapovanam of Alappuzha District, and Poyilkavu of Kozhikode District were analysed using Metagenomics analysis and Illumina sequencing. A total of 30,584, 78,323, and 55,640 reads were obtained from these groves, respectively. Ascomycota constitutes over 96% of the total fungi, making it the most abundant phylum, followed by Mortierellomycota, Basidiomycota, Chytridiomycota, and Rozellomycota. These phyla were subdivided into 20 classes, 40 orders, 83 families, 119 genera, and 135 species, while 1269 OTUs remained unidentified at the species level. Eurotiomycetes predominates the class, while the genus Talaromyces from the family Trichomaceae dominates the genera. Neocarmospora falciformis, Trichoderma lixii, and Candida ethanolic are the most abundant fungal species. Diversity analysis shows that Kollakal Thapovanam is rich in fungal species, while Poyilkavu is rich in biodiversity, with a high degree of dominance. Several species were found only in a particular grove and were absent in others and vice-versa, indicating high fungal specificity. Therefore, fungi have to be preserved in their original habitat. The Principal Coordinate Analysis revealed that each grove is distinct highlighting the importance of preserving the unique diversity of each sacred grove. In conclusion, this research provides valuable information about the soil fungal genera in their natural habitat. It emphasizes the need for more systematic research to understand the actual diversity and ecological role of fungi in sacred groves. This study is the first of its kind to analyse and compare soil fungal diversity in sacred groves using the metagenomics approach.

Keywords: Soil fungi, Sacred groves, Metagenomics, Illumina MiSeq, Diversity analysis, Iringolekavu, Poyilkavu, Kollakalthapovanam

Introduction

Sacred groves, a strategy developed by human beings, have endured in the world for ages as one of the finest instances of traditional conservation methods, preserving nature’s unique biodiversity as such. Sacred groves are mostly found in Africa and Asia, and also exist in Europe and America (Ormsby 2011). India has between 1,00,000 to 1,50,000 sacred groves, primarily located in the Himalayan region, Western and Eastern Ghats, Coastal Region, Central Indian Plateau, and Western Desert (Malhotra 2007). In Kerala, approximately 1500 sacred groves are reported (https://forest.kerala.gov.in/index.php/flora), covering 500 hectares of forest area (Prasad and Mohanan 1995), which contributes to 0.05% of the total forest area of the State (Chandrashekara et al. 1998).

Sacred groves believed to be the adobe of Gods or Goddesses (Khumbongmayum et al. 2005); are the patches of densely vegetated veritable sanctuaries of flora and fauna with perennial water sources. They serve several ecological functions that maintain the ecosystem’s stability (Gadgil 1985). As a result, sacred groves gained a prominent research focus (Gadgil and Vartak 1976), especially in areas related to plant and animal diversity (Tennakoon et al. 2021). However, very few attempts have been made to systematically study the fungal communities within these groves.

Fungi, the second largest group in the world after insects (Hawksworth 1991), remain the least understood group when compared to the other flora and fauna (Perini et al. 2008). Despite their small size, they play several key roles such as decomposers, food sources, pest controllers, producers of antibiotics and hormones, bio-fertilizers, in bioremediation, nutrient and carbon cycling, and improving plant tolerance and health in return for carbon (French 2017). Their true diversity is estimated to be 1.08 million species, but high-throughput sequencing suggests that there may be up to 6.28 million species (Baldrian et al. 2022). Regardless of the difference of opinion, it is apparent that our knowledge about their actual diversity lags behind. In the words of Hawksworth (Hawksworth 1991), “The world’s undescribed fungi can be viewed as a massive potential resource which awaits realization”.

Also, studies on the distribution of soil fungi have mainly focused on agricultural, desert, and saline soils and less is known about the occurrence of fungi in natural soil conditions (Banakar et al. 2012; Schmit and Mueller 2007). It is important to examine soils from localities such as forests, peat bogs, and mountains, which remain untouched by man, to obtain a fair idea of an endemic mycoflora (Ling-Young 1930). In India, particularly in Kerala, limited studies have been conducted on soil fungal diversity regarding habitat, climate, and altitude (Pandey et al. 2006; Satish et al. 2007). Therefore, accurate data on the soil fungi in the natural forests of Kerala is lacking. To address this knowledge gap, we undertake the present work.

Metagenomics technology is a rapidly growing field that provides comprehensive information about the entire genome of microbial communities from environmental samples or original habitats (Chen et al. 1995). It will help to overcome the instability of morphological traits and our inability to mimic fungal growth conditions in laboratory environments which make them even more difficult to identify (Bills and Polishook 1994). The Polymerase chain reaction (PCR) amplification of internal transcribed spacers (ITS) and the D1D2 domain of the large ribosomal subunit using universal primers having barcode sequences (Kurtzman and Robnett 1998), combined with high-throughput sequencing (Lindahl et al. 2013), is a successful method for studying soil mycoflora and analysing multiple samples (Tedersoo et al. 2015; Tonge et al. 2014). However, an integrated lysis method is required to overcome the complexity of isolating fungal DNA from sediments, which is a major limitation of metagenomics analysis of soil fungi (Jiang et al. 2011). However, it has been identified that the ITS1 region, with an average length of 250 bp, is sufficient to identify fungi at the species and genus level (Nilsson et al. 2009).

Therefore, in this study, we aimed to examine the distribution, abundance and diversity of soil fungi in the selected sacred groves of Kerala. To accomplish this, we used internal ITS sequencing on the Illumina MiSeq platform, which is known for its high efficacy in differentiating similar soil samples compared to other next-generation sequencing technologies (Habtom et al. 2017). Additionally, we conducted a phylogenetic analysis of the soil mycoflora from sacred groves to gain further insight into the ecological role and actual diversity of fungi in these areas. This study represents the first report on fungal diversity in sacred groves of Kerala using metagenomics analysis.

Materials and methods

Site description

In the present study, we selected three sacred groves from three different parts of Kerala. The selection was purely based on the area of the sacred grove and permission to enter into the sacred grove. We selected Iringole kavu (S1) (10° 06′ 32.71″ N and 76° 30ʹ 01.44ʺ E) from the Central part of Kerala, Kollakal Thapovanam (S2) (9° 11′ 05.19ʺ N and 76° 27ʹ 41.30ʺ E) from the Southern part of Kerala and Poyilkavu (S3) (11° 24ʹ 31.49ʺ N and 75° 42ʹ 49.37ʺ E) from the Northern part of Kerala.

Iringole Kavu

Iringole kavu is the largest sacred grove in Kerala (https://forest.kerala.gov.in/index.php/flora) with a total area of about 20.234 hectares with a hot and humid climate. In the central part of the grove is the ruling deity, Iringole Kavil Amma, who is considered as Vana Durga. The vegetation type of the grove was reported as West Coast Tropical Evergreen type. But now the vegetation has changed to a Semi-evergreen type (ShanthaKumar et al. 2010). Iringole kavu is associated with two freshwater ponds. The total number of angiosperm species recorded in this sacred grove is about 185 species (Chandrasekhara 2011).

Kollakal Thapovanam

Kollakal Thapovanam is a man-made sacred grove located in the Harippad of Alappuzha district with a total area of 1.214 hectares. The sacred grove is owned by a family and the ruling deity is “Kuriyala Vallichan”, the ancestor of the family. It is associated with 2 freshwater ponds and is about 3 km away from the Sea.

Poyilkavu

Poyilkavu is situated in the Koyilandy of Kozhikode district and is 250 m away from the Seashore. The total area of the grove is about 4.62 hectares with Evergreen-type vegetation. In the central part of the grove is the ruling deity, Poyilkavil Amma, who is considered as Vana Durga. Poyilkavu is associated with a small freshwater pond. The total number of angiosperm species recorded in this sacred grove is about 90, with 14 Endemic species (Chandrashekara et al. 2018).

Soil sampling

Soil samples were collected aseptically from a depth of 0 to 10 cm from various locations within each sacred grove. They were pooled together to form a single sample for each grove. The samples were then stored in polythene bags inside cooler boxes filled with ice blocks and were carried to the laboratory for metagenomics analysis.

DNA extraction

Genomic DNA was extracted from the soil using the DNeasy Power Soil Kit (QIAGEN, Hamburg, Germany) following the manufacturer’s instructions. The sample was prepared as per the manufacturer’s instruction and was homogenised using PowerLyzer 24 Homogenizer. The homogenization with up to 10 cycles of bead beating for as long as 5 min per cycle helps in cell lysis. Cells are lysed by a combination of chemical agents and mechanical shaking introduced at this step. Randomly shaking the beads in the presence of disruption agents will cause the beads to collide with microbial cells and lead to the cells breaking open.

PCR amplification

For PCR, 50 μl reaction mixtures (Emerald Amp GTPCR Master Mix) were prepared according to the manufacturer’s instructions with 10 picomols of forward and reverse primers each. ITS 1–4 genes were amplified using the specific primers [ITS 1 (TCCGTAGGTGAACCTGCGG) and ITS 4 (TCCTCCGCTTATTGATATG)] with barcodes. The PCR amplification was performed using a thermal cycler (BIORAD, USA) with the following protocol. 35 cycles of 95 °C for 3 min, 95 °C for 15 s, 56 °C for 30 s, 72 °C for 30 s, 72 °C for 5 min and hold at 4˚C. The PCR products were analysed by electrophoresis with 2% agarose gel. The samples that produced one bright main strip between 500 bp were selected for further experiments, and bead-based purification was performed.

Library preparation and sequencing

Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit (NEB, USA) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 4.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Then the library was sequenced on an Illumina MiSeq platform, generating 250 bp paired-end reads.

Data processing

The generated paired-end sequences were processed using the QIIME2 tool (version 2022.2.0) (Bolyen et al. 2019). The "Dada2" algorithm (Divisive Amplicon Denoising Algorithm, version 2) (Callahan et al. 2016) was used to filter phiX reads and chimeric sequences in the Illumina amplicon sequence data and to get the number of sequences obtained per sample.

Taxonomic analysis

The pre-processed sequences were clustered in Operational taxonomic units (OTU) using a classifier model trained on the UNITE version 8 (99%) reference database (https://unite.ut.ee/repository.php). The relative taxonomic abundance and count data in all ranks (Kingdom, Phylum, Class, Order, Family, Genus, and Species) were obtained. The clustered heat map was generated at the genus level.

Phylogenetic tree preparation

For the construction of the phylogenetic tree, the pipeline utilizes the mafft program to create a Feature Data [Aligned Sequence] QIIME2 artifact. This involves multiple sequence alignment and masking the alignment to remove highly variable positions that could cause noise in the final tree. Then Fasttree program was used to prepare the tree. The midpoint rooting is applied to position the root of the tree at the midpoint of the longest tip-to-tip distance in the unrooted tree. This information was stored in a phylogeny [Rooted] QIIME 2 artifact. For diversity metrics that count features per sample, a rooted phylogenetic tree is necessary to establish relationships between features.

Soil texture analysis

Soil texture was analysed using a Hydrometer through the Hydrometer method.

Diversity analysis

The diversity analysis of the samples was studied using Alpha diversity and Beta diversity analysis of OTUs.

Alpha diversity

In alpha diversity analysis, the following indices were calculated

Rarefaction Curves (prepared using QIIME2 Software) and Rank Abundance Curves (prepared using QIIME1 software) were performed to examine the species richness and evenness within a single microbial community of each sample.

Beta diversity

Beta diversity analysis was done to observe the similarity of the community structure among different samples. Following Beta diversity indices were calculated (using QIIME2 Software).

  • Unweighted UniFrac distance matrix

  • Weighted UniFrac distance matrix

  • Bray–Curtis distance matrix

  • Jaccard distance matrix

Then Multidimensional Scaling (MDS) analysis was performed. In MDS, Principle Coordinate Analysis (PCoA) is the classical one to examine the similarities and dissimilarities between the samples (Ramette 2007).

Results

Sequence data analysis

The metagenomics sequences of three samples from three sacred groves were deposited at Sequence Read Archive (SRA of National Centre for Biotechnology Information, NCBI) with Bio project number (PRJNA881851) and the Bio-sample accession numbers SAMN30917060, SAMN30917061, SAMN30917062 (Table 1).

Table 1.

“Minimal information about metagenomics sequence” (MIMS) for the NGS sequence data submitted to the SRA database of GenBank in NCBI for Soil of Sacred Groves

Structured comment name
Submitted to Sequence Read Archive (SRA), Accession number: SAMN30917060 Sequence Read Archive (SRA), Accession number: SAMN30917061 Sequence Read Archive (SRA), Accession number: SAMN30917062
Sample name ISG (S1) KSG (S2) PSG (S3)
Project name Metagenomics study of soil fungi from Iringole sacred grove Metagenomics study of soil fungi from Kollakal Thapovanam sacred grove Metagenomics study of soil fungi from Poyilkavu sacred grove
Investigation type Diversity of soil fungi from Iringole Sacred grove Diversity of soil fungi from Kollakal Thapovanam Sacred grove Diversity of soil fungi from Poyilakvu Sacred grove
Environment type Iringole Sacred grove, Ernakulam, Kerala Kollakal Thapovanam Sacred grove, Alappuzha, Kerala Poyilakavu Sacred grove, Kozhikode, Kerala
Geographical location name India India India
Collection date 10–04-2022 17–04-2022 07–05-2022
Latitude and Longitude 10°06′ 32.71ʺ N 76°30′ 01.44ʺ E

9°11′ 05.19″ N

76°27′ 41.30ʺ E

 11°24ʹ31.49ʺ N 75°42ʹ49.37ʺ E
Elevation 37 m 2 m 5 m
Material Soil Soil Soil
Depth 0 to 10 cm 0 to 10 cm 0 to 10 cm
Organism Soil fungi Soil fungi Soil fungi
Biome Sacred grove Sacred grove Sacred grove
Material Soil Soil Soil
Sequencing Method Illumina MiSeq Illumina MiSeq Illumina MiSeq
Library Strategy ITS 124 Amplicon ITS 124 Amplicon ITS 124 Amplicon
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A total of 110,426, 283,208 and 192,866 reads were obtained from the soil samples taken from Iringole kavu, Kollakal Thapovanam and Poyilkavu respectively. Among these, 55,213 reads were paired-end reads in S1, while 141,604 and 96,433 reads were paired-end reads in S2 and S3, respectively. These paired-end reads are in the format of FASTq files with file names S1_R1.fastq.gz, S2_R1.fastq.gz, and S3_R1.fastq.gz, and are 250 bp in length. The average GC% for S1, S2, and S3 were 58.55%, 58.56%, and 58.68%, respectively. All fastq files were imported into the QIIME2 software for further analysis. The "Dada2" method was utilized to denoise the sequences and to better discriminate between true sequence diversity and sequencing errors. The summary of the denoising statistics is given in Table 2.

Table 2.

Summary of denoising statistics

Input Filtered Input passed filter % Denoised Merged Input merged % Non-chimeric Input non-chimeric %
S1 55,213 45,126 81.73 44,365 30,854 55.88 30,854 55.88
S2 1,41,604 1,18,948 84 1,17,804 78,365 55.34 78,323 55.31
S3 96,433 85,634 88.8 84,740 55,640 57.7 55,640 57.7
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After the paired-end reads were denoised, we obtained a total of 30,854 reads for S1, 78,323 reads for S2, and 55,640 reads for S3. These reads were then clustered into Operational Taxonomic Units (OTUs) using a classifier model trained on UNITE version 8. Once the OTUs were identified, they were classified into Phylum, Class, Order, Family, Genus, and Species for each sample (Fig. 1).

Fig. 1.

Fig. 1

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The chart representing the number of OTUs obtained at each classification level for samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Taxonomy analysis

The taxonomic hierarchy of OTU was analysed using a Krona plot (Figs. 2, 3, 4). The categories Kingdom, Phylum, Order, Class, Family and Genus were selected. Less abundant and unresolved taxa, grouped as unidentified, were listed outside the chart with their relative abundance. The Krona plot revealed that Ascomycota is the most abundant phylum in all the three sacred groves, accounting for about 97%, 98% and 96% of the total OTU of S1, S2, and S3, respectively. Other phyla observed were Mortierellomycota, Basidiomycota, Chytridiomycota, and Rozellomycota. The composition and abundance distributions at the six levels of classification of each sample were obtained and were given in (Figs. 5, 6).

Fig. 2.

Fig. 2

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Krona plot showing the taxonomic hierarchy of OTU from soil sample of Iringole sacred grove (S1)

Fig. 3.

Fig. 3

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Krona plot showing the taxonomic hierarchy of OTU from soil sample of Kollakal Thapovanam sacred grove (S2)

Fig. 4.

Fig. 4

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Krona plot showing the taxonomic hierarchy of OTU from soil sample of Poyilkavu sacred grove (S3)

Fig. 5.

Fig. 5

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Taxonomic composition and abundance distribution of OTU at Phylum, Class and Order levels for samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Fig. 6.

Fig. 6

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Taxonomic composition and abundance distribution of OTU at Family, Genus and Species levels for samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

In Iringole sacred grove, Ascomycota is the most abundant phyla followed by Mortierellomycota (1.7%), Basidiomycota (0.6%), Chytridiomycota (0.2%), and Rozellomycota. These phyla were subdivided into 12 classes, 22 orders, 42 families, 53 genera, and 56 species. In Kollakal Thapovanam, Ascomycota is followed by Basidiomycota (2%), Mortierellomycota (0.4%), Rozellomycota (0.3%) and Chytridiomycota (0.01%). These phyla were subdivided into 17 classes, 33 orders, 60 families, 81 genera, and 88 species. In Poyilkavu, Ascomycota is the dominant phyla followed by Basidiomycota (3%), Mortierellomycota (0.5%), Rozellomycota (0.04%) and Chytridiomycota (0.02%). These phyla were subdivided into 15 classes, 29 orders, 50 families, 69 genera, and 67 species. Taxonomically unresolved sequences were grouped under unidentified Class, Family, Order, Genera, and Species.

On the class level, Eurotiomycetes belonging to phylum Ascomycota is the most prominent class in all the three sacred groves, accounting for about 93% of the OTU in S1, 91% of the OTU in S2 and 89% of the OTU in S3. Sordariomycetes of Ascomycota is the second most abundant class. Mortierellomycetes (2%) of the phylum Mortierellomycota, Saccharomycetes (0.6%) and Dothidiomycetes (0.5%) of the phylum Ascomycota were the other dominant classes found in Iringole kavu. In Kollakal Thapovanam, Saccharomycetes (1%) of Ascomycota, Tremellomycetes (1%) of Basidiomycota and Dothidiomycetes (0.7%) of the phylum Ascomycota were the other dominating classes. In Poyilkavu, Agaricomycetes (2%) of Basidiomycota, Dothidiomycetes (0.6%) of Ascomycota and Mortierellomycetes (0.5%) of Mortierellomycota were the dominating classes.

Out of the 20 classes identified, Kollakal Thapovanam has the highest number of classes (17). Class Rhizophydiomycetes were seen only in Iringole kavu, Archaeorhizomycetes were seen only in Poyilkavu, whereas Ustilaginomycetes, Exobasidiomycetes, Rozellomycotina cls Incertae sedis and Orbilliomycetes were seen only in Kollakal Thapovanam. Class Geminibasidiomycetes was seen only in Iringole kavu and Poyilkavu. Leotiomycetes, Microbotryomycetes and Agaricostilbomycetes were seen only in Kollakal Thapovanam and Poyilkavu (Table 3).

Table 3.

Fungal classes identified from samples S1, S2 and S3

Class S1 S2 S3
Eurotiomycetes 28,240 69,722 48,935
Sordariomycetes 1018 3720 3564
Mortierellomycetes 538 353 295
Saccharomycetes 249 983 172
Dothideomycetes 149 625 364
Tremellomycetes 93 907 127
Pezizomycetes 41 18 75
Agaricomycetes 41 343 1406
Cystobasidiomycetes 15 72 6
Wallemiomycetes 15 28 11
Geminibasidiomycetes 7 0 51
Leotiomycetes 0 6 2
Microbotryomycetes 0 24 4
Agaricostilbomycetes 0 3 2
Rhizophydiomycetes 40 0 0
Ustilaginomycetes 0 5 0
Rozellomycotina_cls_Incertae_sedis 0 35 0
Exobasidiomycetes 0 8 0
Orbiliomycetes 0 68 0
Archaeorhizomycetes 0 0 7
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Based on the number of representative OTU, Eurotiales (Class Eurotiomycetes) is the most abundant order in all the three sacred groves with 28,228 OTU in S1, 69,687 OTU in S2 and 48,907 OTU in S3 followed by Hypocreales. Mortierellales, Saccharomycetales and Sordariales are the other dominating orders found in Iringole kavu whereas Saccharomycetales, Tremellales and Capnodiales are the other dominating orders in Kollakal Thapovanam. In Poyilkavu, Agaricales, Boletales and Mortierellales became the other dominating orders.

A total of 40 orders were identified, and Kollakal Thapovanam had the highest number of orders with 33. Seven orders, including Leucosporidiales, Ustilaginales, Doassansiales, Erythrobasidiales, Botryosphaeriales, Dothideales, and Orbiliales were seen only in Kollakal Thapovanam. Meanwhile, Rhizophydiales and Oxygenales were found only in Iringole kavu. Cantharellales, Trichosphaeriales, Archaeorhizomycetales and Trechisporales were found only in Poyilkavu.

Family Trichomaceae was the most abundant family in all three sacred groves, with 28,145 OTU in S1, 69,432 OTU in S2 and 48,509 OTU in S3. Mortierellaceae, Necteriaceae, and Incertae sedis of the Pezizales family were the other dominating families in Iringole kavu. Meanwhile, Nectericaceae and Hypocreaceae became the other dominating families in Kollakal Thapovanam and Poyilkavu.

Out of 83 families identified, Kollakal Thapovanam had the highest number of families (60). 26 families were common to all the sacred groves. 21 families were seen only in Kollakal Thapovanam, 11 families were found only in Poyilkavu, and 9 families were found only in Iringole kavu.

At the genus level, Talaromyces of Trichocomaceae family predominated the sample with 28,134 OTU in S1, 9,427 OTU in S2 and 48,493 OTU in S3. A Barplot was used to show the abundance of each genus in the sample (Fig. 7), which clearly showed the predominance of the Talaromyces genus over the others. Other abundant genera in S1 were Mortierella, Neocarmospora, Acremonium and Candida of Incertae sedis. In S2 and S3, following Talaromyces, Neocarmospora and Trichoderma were abundant.

Fig.7.

Fig.7

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Barplot showing abundance of each Genus in the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

A Venn diagram was made at the genus level (Fig. 8), showing that 30 genera were common in all three sacred groves. Four genera were found only in S1 and S2, six genera were found only in S1 and S3 whereas 16 genera were found only in S2 and S3. 32 genera were seen only in S2 whereas 14 genera were seen only in S1, and 18 genera only in S3. A Heat map (Fig. 9 was prepared at the genus level, where each row corresponds to a genus (OTU), and each column corresponds to a sample. The higher the relative abundance of an OTU in a sample, the more intense the colour at the corresponding position in the Heat map. Here, yellow represents more abundance, while blue represents less abundance.

Fig. 8.

Fig. 8

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Venn diagram showing the similarity and dissimilarity at the Genus level between samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Fig. 9.

Fig. 9

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Clustered Heat map at Genus level showing the relative abundance of OTU in the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

A total of 135 species were identified from the sacred groves. In that Neocarmospora falciformis, Trichoderma lixii and Candida ethanolica were some of the common and abundant species found in all the sacred groves. But Mortierella minutissima is the species that dominates in Iringole kavu which is absent in other sacred groves. However, 1269 OTUs remain unidentified at the species level.

Out of 135 species identified, Kollakal Thapovanam had the highest number of species (88). 25 species were common to all the sacred groves. 44 species were seen only in Kollakal Thapovanam, 20 species were found only in Poyilkavu, and another 20 species were found only in Iringole kavu.

Soil texture analysis

Soil texture was analysed using the Hydrometer method. The soil texture of each grove has turned out to be different. Iringole kavu has Loamy Sand soil whereas the soil texture of Kollakal Thapovanam is Sand and the soil texture of Poyilkavu is Gravelly Clay Loam.

Diversity analysis

A phylogenetic tree was prepared using the FastTree program, which computed phylogenetically-based alpha diversity metrics. The alpha diversity analysis estimates the abundance and diversity of species in environmental communities, as well as the number of species in the fungal community. The alpha diversity indices were calculated and are given in Table 4.

Table 4.

Alpha diversity Indices among the sacred groves

Chao 1 Observed features Shannon index Simpson index
Iringole kavu 133 133 1.408152694 0.289899993
Kollakal Thapovanam 235 235 1.730943685 0.324326373
Poyilkavu 179 179 1.816606482 0.356734908
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The richness estimators, such as Chao 1 and Observed features, revealed that Kollakal Thapovanam has the highest species richness, followed by Poyilkavu and Iringole kavu. This indicates that Kollakal Thapovanam is richer in fungal species. However, according to the Shannon and Simpson indices, Poyilkavu exhibits higher values. Therefore, based on the Shannon index, Poyilkavu has a rich biodiversity compared with the other two sacred groves. According to the Simpson index, Poyilkavu has a higher value, indicating higher dominance.

The Rarefaction curve of Observed features, and Shannon index, and the Rank Abundance curve were plotted (Fig. 10). In the Rarefaction curve of Observed features, the plateauing of the curves revealed that the generated sequence reads and determined OTUs were sufficient to capture the fungal diversity of these three samples. The Rarefaction curve of the Shannon index indicates that with the increase in sample size, the rate of new species also increases. The Rank Abundance curve reflects an even distribution of fungal taxa in all three sacred groves due to the shallow gradient of the curve.

Fig. 10.

Fig. 10

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Alpha Diversity analysis of the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu). A Rarefaction curve of Observed features. B Shannon Rarefaction curve. C Rank abundance curve

The Beta Diversity analysis was calculated to measure the in-between sample diversity and is given in Table 5. The Bray–Curtis dissimilarity index for all three sacred groves falls between 0 and 1, indicating that they share common species but not all are common. The Jaccard similarity index analysis shows that Iringole kavu and Kollakal Thapovanam are more similar, and Iringole kavu and Poyilkavu are the less similar ones when compared with others.

Table 5.

Beta Diversity Indices among the sacred groves

graphic file with name 13205_2024_3932_Tab5_HTML.jpg

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The PCoA analysis of Weighted UniFrac (Fig. 11), Unweighted UniFrac (Fig. 12), Jaccard (Fig. 13), and Bray–Curtis (Fig. 14) reveals that the objects were not close to each other, and hence these three sacred groves were very much different from one another.

Fig. 11.

Fig. 11

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Beta Diversity Analysis—Weighted UniFrac PCoA Analysis of the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Fig. 12.

Fig. 12

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Beta Diversity Analysis—Unweighted UniFrac Analysis of the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Fig. 13.

Fig. 13

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Beta Diversity Analysis—Jaccard PCoA Analysis of the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Fig. 14.

Fig. 14

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Beta Diversity Analysis—Bray–Curtis PCoA Analysis of the samples S1 (Iringole kavu), S2 (Kollakal Thapovanam) and S3 (Poyilkavu)

Discussion

The metagenomics approach for analysing and comparing the composition and diversity of soil fungi of sacred groves in Kerala became the first report of its kind.

The metagenomics analysis of soil samples using ITS-124 Amplicon Illumina Sequencing and the feature taxonomic classification resulted in five phyla. The study found that soil fungal community structure predominately consists of phylum Ascomycota followed by Basidiomycota, Mortierellomycota, Chytridiomycota, and Rozellomycota. However, in Iringole kavu, Mortierellomycota was the second most common phyla which was consistent with the studies of Haldar and Nazareth (2019), Moussa et al. (2017), Baeza et al. (2017), Simoes et al. (2015) and Yadav et al. (2015). Ascomycota and Basidiomycota play important roles in maintaining ecosystem stability (Challacombe et al. 2019; Hanson et al. 2008) and Mortierellomycota fungi are known to promote plant growth (Ozimek and Hanaka 2020). The most prominent class in the soil samples of sacred groves was class Eurotiomycetes of phylum Ascomycota, followed by Sordariomycetes. Our findings corroborate the earlier findings of Hedeler et al. (2007) and Moussa et al. (2017).

Class Archaeorhizomycetes identified from Poyilkavu with very low relative abundance provide an additional piece of knowledge to their occurrence, which correlates with the work of Choma et al. (2016). Archaeorhizomycetes was one of the ancient classes of soil fungi (Rosling et al. 2011) and a keystone taxon that plays a non-negligible role in the soil fungal community (Choma et al. 2016).

Genus Talaromyces of family Trichomaceae predominates in all three soil samples, correlated with the study report of Han et al. (2020). However, earlier soil studies reported that Penicillium was the most abundant genus (Mohamed and Nair 2016; Sankaran and Balasundaran 2000) and this may be the misidentification of the genus Talaromyces as Penicillium, as the earlier contains the teleomorph of the genus Penicillium (Pitt 1979). Talaromyces species is a biocontrol agent against plant pathogenic fungi (Dethoup et al. 2007) and is an anti-insectant (Nicoletti and Becchimanzi 2021), the compounds isolated from the secondary metabolites of Talaromyces have anti-inflammatory, antibacterial and antitumor activities. Hence, can say that a high number of Talaromyces is positively influencing the plant diversity of sacred groves. The taxonomically unresolved sequences were grouped as unidentified and represented approximately 9.4% of the total OTU. But Nagy et al. (2011) reported that a lag in type strain and specimen sequencing may result in an unusually high number of unidentifiable MOTUs, at least in some fungal groups than a high number of undescribed species.

Taxonomic classification of OTU reveals that the abundance and composition of soil fungi vary in each grove even though the phyla observed were similar. The Bar plot demonstrated the dominance of the genus Talaromyces in all groves, but the genus composition was different. 32 genera were unique to Kollakal Thapovanam, 18 were unique to Poyilkavu, and 14 were unique to Iringole kavu.

The diversity analysis revealed that Kollakal Thapovanam had relatively more fungal species, whereas Poyilkavu had greater biodiversity and showed high dominance. Furthermore, an increase in sample size resulted in an increase in the new species rate, and the chance of obtaining the same genus at a random selection was medium. Based on the comparative analysis, it was observed that certain species were found exclusively in specific groves, despite the presence of common species in sacred groves. The groves, Kollakal Thapovanam and Iringole Kavu were found to be more similar to each other. At the same time, PCoA revealed that each grove was distinct.

The work revealed that certain groups of fungi are highly specific to a particular sacred grove and are absent in others, and vice versa. This indicates that fungi have a high degree of specificity in occurrence. Previous research has suggested that differences in soil texture and vegetation among the sacred groves affect fungal diversity (Griffin 1963; Khalid et al. 2006). Therefore, it is established that the variation in fungal diversity and specificity in occurrence may be due to the difference in soil texture and vegetation among the three sacred groves. Moreover, the study found that minor changes in the habitat can cause specific fungi to become extinct consistent with Chen et al. (2023) research that indicates the distribution of mycobiome is influenced by habitat specificity. As a result, our research highlights the significance of conserving each sacred grove in its original form.

Conclusion

The study concludes that the sacred groves in Kerala, India have a unique and distinct fungal diversity, despite some similarities, indicating that they are a rich repository of soil fungi. The vegetation, soil texture and composition of fungi in each grove were found to be different, and small changes in the habitat of specific fungi can lead to their extinction. This emphasizes the importance of preserving the purity of every sacred grove, as they are miniature forests with rich biodiversity. Moreover, this work reveals that the actual number of microfungi remains unknown. The present study provides prior information that can help to reveal the undiscovered genera of the soil ecosystem. Therefore, further research with continuous monitoring of sacred groves is necessary to identify the actual diversity of fungi and their ecological role. The work points to the need for functional metagenomics in future to identify the unknown species found in this study.

Acknowledgements

The author’s acknowledged the OmicsGen LifeSciences Pvt Ltd Labs for the use of Illumina MiSeq and DST-FIST sponsored Lab, Department of Botany and Library, St. Thomas College (Autonomous), Thrissur for their assistance.

Author contributions

All authors contributed to the study’s conception and design. Material preparation, data collection and analysis were performed by KN. The first draft of the manuscript was written by KN and all authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.

Funding

The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Data availability

The datasets generated during and /or analysed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The Authors have no conflict of interests to declare that are relevant to the content of this article.

Ethical approval and Consent to participate

Not applicable as this research does not involve any human or animal trials.

Consent for publication

The submission of this paper for publication in the journal “3 Biotech” has been approved by all the authors and all authors read and approved the Manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and /or analysed during the current study are available from the corresponding author upon reasonable request.

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