Prokaryotic diversity of tropical coastal sand dunes ecosystem using metagenomics

Abstract

Coastal sand dunes (CSDs), unique, stressed and hostile habitats act as a barrier between marine and terrestrial ecosystems. CSDs are stressed in terms of nutrition and fluctuating physio-chemical conditions. CSD is classified into several types, each of which presents different challenges for life forms. This study focuses on exploring bacterial and archaeal diversity and community structure in four CSD namely, Embryo, Fore, Gray, and Mature dunes of Keri beach, Goa along the west coast of India. The study was carried out using Next Generation Sequencing of hypervariable V3–V4 regions of the 16S rRNA gene using Illumina HiSeq platform. The present study hypothesizes that the prokaryotic communities at each dune may be different and could have different role in the ecosystem. The NGS for Embryo, Fore, Gray, and Mature dunes gave 1,045,447, 1,451,753, 1,321,867, and 1,537,758 paired-end reads, respectively, out of which 54,500, 50,032, 37,819, and 111,186 were retained through various quality filtrations. A total of 74, 63, 65, and 65% of OTUs, respectively, remained unknown at the species level. The highest bacterial and archaeal abundance was reported from Mature and Embryo dunes, respectively. Phylum Actinobacteria dominated the Embryo, Fore, and Mature dunes, whereas phylum Proteobacteria was the dominant in the Gray dune. Streptomyces was predominant in overall CSD followed by Bacillus, Acidobacterium, and Kouleothrix. The commonly and exclusively found members in each dune are cataloged. The highest species dominance, diversity, species richness, and abundance were observed in Embryo, Fore, Gray, and Mature dunes, respectively. The present study clearly elucidates that each dune has a distinct microbial community structure.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02809-5.

Introduction

Exploring microbial communities is a crucial factor in studying the various roles played by these in the ecosystem. Coastal sand dunes (CSD), lie at the intersection of marine and terrestrial ecosystems, formed when windblown sand is trapped by beach grasses over many years and dynamically interact with tides (Poyyamoli et al. 2012). CSDs are one of the least studied ecosystems. They are specialized habitats characterized by sparse vegetation and subjected to the influence of salty sea mists. CSDs provide an interesting niche to study microbial diversity. These occupy approximately 8% of the total earth’s surface and are one of the most taxonomically rich and productive ecosystems on earth (Ramarajan and Murugesan 2014). CSD has been identified as one of the most important biogeographical habitats of India (Rodgers and Panwar 1988). India has about 7516 km long coastline covering 2.1 Million km² and nine states (Khoshoo 1996). Goa is the smallest state of India and has a stretch of about 22.62 km of sand dunes facing the Arabian Sea. CSDs are known as “natural barriers” which protects the inland from harsh winds and waves. These also provide a resourceful habitat to a variety of flora and fauna. CSDs are stressed and extreme habitats in terms of sparse nutrient availability, low moisture content, regular salt spray, intense UV rays, constant sand burial, and fluctuations in temperature and pH (Aureen et al. 2010). These extremely harsh conditions limit the growth of various species. Despite these disturbances and nutrient deficiencies, diverse flora, fauna, and microorganisms have adapted to CSD habitats (Nayak et al. 2013; Muthukumar and Samuel 2011; Desai 2005; Aureen et al. 2010; Gaonkar et al. 2012). These can tolerate strong wind, salt spray, sand burial, desiccation, constant sand shifting, little to no organic matter, and low water retention capacity of sand.

Microorganisms inhabiting CSD are subjected to several environmental fluctuations that affect their growth and community structure. CSD has a predominance of leguminous plants such as Casuarina and Canavalia, possessing nodules that form a symbiotic association with N₂-fixing bacteria serving as N₂-fixers (Arun and Sridhar 2004; Seena et al. 2007). Understanding the stress tolerance in nature and possible applications of stress-tolerant genes in agriculture is immensely important. Microorganisms such as Rhizobia, Mycorrhizae, and diazotrophs adapting to CSD’s extreme environmental conditions require special attention. Microorganisms from the CSD ecosystem are known to produce various secondary metabolites that are bioactive and are significant in several industries including agriculture and pharmaceuticals. Numerous species of Rhizobium, Pseudomonas, and Bacillus, associated with the roots of plants in CSD, are capable of producing compounds such as indole acetic acid and siderophores, which are responsible for plant growth promotion (Arun and Sridhar 2004). Further, Pseudomonas sp., Bacillus sp., and Streptomyces sp. obtained from CSD have antagonistic activity towards plant pathogens such as Rhizoctonia solani and Fusarium oxysporum (Shin et al. 2007). Many halophilic and alkaliphilic bacteria from CSD produce extracellular proteases, cellulases, amylases, xylanases, chitinases, pectinases, and tannases which are vital in several industries (Sangeetha et al. 2012; Kedar et al. 2014).

CSDs are also impacted by various anthropogenic activities and increase in tourism. Thus causes immense pollution and contamination, which ultimately, disturbs the flora and fauna of CSD. Oil spillage in the sea is a major problem faced by many countries, subsequently leading to tarball deposition on CSD. The state of Goa is facing tarball pollution (Suneel et al. 2015). These tarballs contain high molecular weight n-alkanes and polycyclic aromatic hydrocarbons which are often been reported from coastal areas across the world (Warnock et al. 2015). These problems influence microbial diversity, and ultimately the overall biological diversity. Therefore, exploring the microbial diversity of CSD is crucial particularly along the west coast of India. Despite CSD’s importance in the ecosystem, there are a very few studies exploring its microbial diversity.

CSD is classified into four dunes based on the distance from the coastline; pH and vegetation present (Desai 2005). Embryo dune lies parallel to the ocean, consisting of pH 8.5 and mostly devoid of any kind of vegetation. Fore dune having the pH 7.5–8 is located just above the high tide line and runs parallel to the first beach ridge. Pioneer grasses, Ammophila arenaria and Ipomea pes-caprae are characteristics of Fore dune. Yellow dune is characterized by having more humus and pH 7.5. Commonly found plant species in Yellow dune are Calystegia soldanella, Eryngium maritimum, and Carex arenaria. Gray dune is located 50–100 m away from the Embryo dune and lies toward the land side. The pH of this dune ranged from 5 to 6 and vegetation includes various shrubs. Mature dune is found several hundred meters away from the shore and consists of climax vegetation of oaks and pines. The pH of Mature dune is acidic, ranging from 4 to 5, due to the presence of a high amount of humus. However, the types of dunes present and the distance between these may vary from one CSD to another. The vegetation of CSD plays a pivotal role in the ecosystem by building a rhizome system that releases the root exudates, thereby supporting the microbiome and also helps in binding the sand particles together (Muthukumar and Samuel 2011).

There have been reports wherein scientists have studied the physiological, geological, and restoration aspects of CSD (Mascarenhas and Jayakumar 2008). However, very few investigations have been carried out on the bacterial communities of CSD. From the ecological point of view, bacterial communities at such extreme and stressed conditions are important. Therefore, exploring such habitats will facilitate tremendous knowledge of the bacteria and their potential in various fields. Few attempts have been made over the last decade to understand the bacterial community structure at CSD using culture-independent and culture-dependent approaches. Bacterial diversity of CSD at the Mediterranean Sea stated that diversity is highest at inland and lowest at the seashore in both, wet and dry seasons (Wasserstrom et al. 2017). However, this study does not explore the diversity at each dune and does not emphasize the abundance, richness, evenness, and the members found. Lin et al. (2014) studied bacterial communities at CSD using a culture-independent technique indicated the predominance of Acidobacteria and Proteobacteria. Earlier reports on culture-based isolates obtained from CSD of Goa, India, have shown the presence of pigmented bacteria including Pseudomonas aeruginosa, Microbacterium arborescens, Bacillus marisflavi, Bacillus magaterium, Alcanivorax sp., and Marinobacter (Gaonkar et al. 2012; Godinho and Bhosle 2013; Prabhu et al. 2018; Nayak et al. 2013; Shinde et al. 2020). These strains have shown tremendous beneficial attributes such as polyhydroxyalkanoates production, hydrocarbons/tarball degradation, bioremediation, and plant growth promotion. Therefore, the current study proposes CSD as a potential source for availing beneficial bacterial strains. The present work focused on studying bacterial and archaeal communities, diversity, abundance, and exclusiveness at four dunes namely Embryo, Fore, Gray, and Mature dunes of CSD of Keri beach, Goa, India, using Rapid Illumina HiSeq 2500 Next Generation Sequencing (NGS).

Materials and methods

Sampling

A total of thirteen sand samples from Embryo, Fore, Gray, and Mature dunes of CSD were collected from Keri beach, Goa, India (15° 42′ 39.84″ N, 73° 41′ 41.29″ E), on 25th January 2015, during low tide. CSD of Keri beach covers about 21,416 sq. meters and has approximately 374 m length and 78 m width. The height of the sand dune ranged from 2.5 to 4 m. The sand samples were collected in sterile zip lock bags and stored at – 20 °C until further processing. The temperature of the sand was recorded on-site using a thermometer. The pH of the samples was recorded using a pH meter (Eutech Instruments, pH 700), by preparing a 10% suspension of each sample in distilled water. One non-rhizosphere sand samples from Embryo dune (S1) and one rhizosphere sand samples from Mature (S4) dune were collected. Composite sand samples of Fore and Gray dunes were prepared separately by thoroughly mixing the K2–K6 samples (S2) and K7–K12 (S3) samples, respectively. The details about the sand samples are provided in Table 1.

Table 1.

Characteristics of samples collected from CSD of Keri beach, Goa, India

S. no.	Sample	Dune	Zone	Temp. (°C)	pH	Sample composite
1	Keri 1 (K1)	Embryo (E)	NR	24.0	6.38	S1
2	Keri 2 (K2)	Fore (F)	NR	23.0	6.70	S2
3	Keri 3 (K3)	Fore (F)	Casuarina equisetifolia*	28.0	6.15
4	Keri 4 (K4)	Fore (F)	Ipomoe pes-caprae*	27.8	6.23
5	Keri 5 (K5)	Fore (F)	Casuarina equisetifolia*	27.0	5.59
6	Keri 6 (K6)	Fore (F)	Launaea nudicaulis*	27.2	6.17
7	Keri 7 (K7)	Gray (G)	NR	29.0	5.79	S3
8	Keri 8 (K8)	Gray (G)	NR	29.0	5.61
9	Keri 9 (K9)	Gray (G)	Gomphrena globosa*	29.0	5.67
10	Keri 10 (K10)	Gray (G)	Spinifex littoreus*	27.0	6.12
11	Keri 11 (K11)	Gray (G)	Acrocephalus capitatus*	28.0	6.25
12	Keri 12 (K12)	Gray (G)	Clerodendrum inerme*	27.0	5.83
13	Keri 13 (K13)	Mature (M)	Anacardium occidentale*	28.2	5.71	S4

NR non-rhizosphere, Temp temperature

*Rhizosphere

Metagenome DNA extraction, PCR amplification, library preparation, and 16S rRNA Illumina Hiseq sequencing

1 g of each sand sample was subjected to DNA extraction using the DNeasy PowerSoil kit (Qiagen). The concentration of the isolated DNA was evaluated using NanoDrop and Qubit, whereas quality was checked by 1% agarose gel electrophoresis (MultiSUB horizontal Gel system-Cleaver Scientific). All four CSD samples were analyzed in three replicates. The hypervariable V3–V4 regions of the 16S rRNA gene were amplified using primers 341F [5’CCTACGGGNBGCASCAG3’] and 805R [5’GACTACNVGGGTATCTAATCC3’] (Qiu et al. 2020) followed by library preparation for paired-end amplicon using NEBNext Ultra DNA Library preparation kit (New England Laboratories, USA). The amplified libraries were purified through bead purification using XP beads (Agencourt Ampure). The fluorometric quantifications were performed for the purified product using Qubit™ 2.0 Fluorometer (Thermo Fisher Scientific, USA). The libraries were sequenced using Rapid Illumina HiSeq 2500 with 2 × 250 bp reads to get paired-end sequences at the M/s AgriGenome Labs Pvt Ltd., Kerala, India. An amount of 10–20 pM of the purified amplified products was loaded onto the Illumina platform. The raw Illumina paired-end reads of Embryo, Fore, Gray, and Mature dunes were submitted in NCBI’s Sequenced Read Archive (SRA) under the accession SRR 12,031,846, SRR 12,031,952, SRR 12,032,010, and SRR 12,032,019, respectively, and are associated with BioProject PRJNA639930.

Bioinformatics analysis

Raw sequences of the V3–V4 regions of the 16S rRNA gene obtained from the Illumina HiSeq platform were processed via Quantitative Insights into Microbial Ecology (QIIME 1.7.0) software. Raw sequences were trimmed to remove primers. The sequences obtained from NGS data were assembled using the De-novo assembly pipeline to give contigs from two paired-end reads using the de Bruijn graph (Zerbino and Birney 2008). Quality control was checked with the Phred quality score (≥ Q30) and used for the clustering of operational-taxonomic units (OTUs). OTU filtering was done with more than five reads. For each sample, highly similar sequence reads (about 97% sequence similarity) were clustered together and a consensus sequence was generated. A consensus sequence was represented as a single bacterial and archaeal sequence in a sample. The number of sequences used to build the consensus formed the basis for quantification of the bacterial and archaeal species. Each consensus sequence was given a taxonomy-based similarity (< 97% similarity in 16S rRNA gene sequence) with the existing bacterial and archaeal species. The taxonomical classification of different OTUs was done based on the SILVA database (Quast et al. 2012).

Bacterial and archaeal community analyses

The relative abundance of bacteria and archaea on phyla, classes, orders, and genera level taxon in each dune were calculated. Species dominance, alpha diversity indices (Simpson and Shannon), Pielou’s evenness (J’), Margalef’s species richness, observed-species indices, rarefaction, and Chao1 curves were calculated using Paleontological statistical software [PAST version 3.25] (Hammer et al. 2001). Beta diversity among the four dunes was calculated using the formula S/$\overline{\alpha }$-1, where, S is the total number of species and $\overline{\alpha }$ is the average number of species (Whittaker 1960). Venn diagrams were constructed to study the exclusively and commonly found phyla, classes, genera, and species among the four dunes. Heatmaps on genera and phyla level were plotted in Rstudio version 3.5.1. using heatmaply package. A UPGMA tree based on the unweighted uniFrac approach from raw consensus sequences of four samples were constructed using Rstudio and Phyloseq package.

Results

Sample analysis

Thirteen sand samples were collected from different zones of CSD of Keri beach, Goa, India (Table 1, Online Resource 1). The temperature and pH recorded for the samples were found to be in the range of 23 °C to 29 °C and 5.59 to 6.70, respectively (Table 1).

Bacterial and archaeal communities at CSD (diversity and richness of OTUs)

A total of 1,045,447, 1,451,753, 1,321,867, and 1,537,758 paired-end reads were obtained from Embryo, Fore, Gray, and Mature dunes, respectively, using Rapid Illumina Hiseq sequencing (Table 2) with a mean read length of 250 bases. The Phred score distribution (≥ Q30) of the paired-end reads derived from the samples of Embryo, Fore, Gray, and Mature dunes were 95.81, 96.15, 95.31, and 93.14%, respectively. GC content distributions of reads from these four samples were 57.24, 56.88, 56.16, and 56.11%, respectively. After quality filtration using conserved region filter, mismatch filter, and chimeras filter, the Hiseq pre-processed sequencing yields were 56,291, 51,731, 39,083, and 126,477 from Embryo, Fore, Gray, and Mature dunes, respectively. A summary of the sequence reads passed through different filters are given in Table 2. After the removal of singletons, total filtered OTUs reads from Embryo, Fore, Gray, and Mature dunes were 54,500, 50,032, 37,819, and 111,186, respectively (Table 2), indicating that Embryo, Fore, and Gray dunes retained 96% whereas Mature dune retained 87.9% of sequences. Out of total OTUs, 77.55% for Embryo, 76.1% for Fore, 76.34% for Gray, and 83.39% for Mature dune, were taxonomically identified (clustered at 97% similarity level) using the SILVA database. The sequences which did not show alignment with the sequences in the taxonomical database were considered unclassified. From all the four dunes, approximately 1% of the total OTUs were unclassified at the domain level. From Embryo dune, bacteria were 93% OTUs and archaea were 6% OTUs (Table 2). From Fore dune, 95% of OTUs were bacteria and 4% of OTUs were archaea. From Gray dune, 96% OTUs and 3% of OTUs were bacteria and archaea, respectively. From Mature dune, 97% of OTUs were bacteria and 2% of OTUs were archaea. Archaea are found to be highest in Embryo dune. A total of 33 phyla, 62 classes, 553 genera, and 956 species were identified from CSD. About 62–75% of total OTUs were unclassified at the species level. Embryo and Gray dunes comprised 27 bacterial phyla, Fore dune contained 22, and Mature dune comprised 21 bacterial phyla. Two archaeal phyla (Euryarchaeota and Thaumarchaeota) were found in all the four dunes. Phyla, classes, orders, family, genera, and species comprising less than 1% of total OTUs were considered as minor groups. The distribution of total bacterial and archaeal OTUs, based on their known, “Candidatus”, and unknown status from each taxonomical level is given in Table 3. “Candidatus” numbers were seen hiked on the genus and species level of taxonomy in all four dunes. The known bacteria and archaea decreased as the taxonomical level increased; conversely, the unknown members increased.

Table 2.

Metagenome sequence analysis of composite sand samples of Embryo, Fore, Gray, and Mature dunes

Sample	Dune	Qubit conc (ng/μl)	Nanodrop conc (ng/μl)	260/280 ratio	Total elute conc. (ng)	Total paired-end reads	Total consensus seq	Chimeric sequences	Pre-processed reads	Total filtered OTUs	Taxonomically identified OTU		Unclassified OTUs
											Bacteria	Archaea
S1	Embryo	13.1	26.4	1.76	55.6	1,045,447	562,361	227,172	56,291	54,500	50,491	3,413	596
S2	Fore	23.4	23.9	1.99	84.0	1,451,753	758,249	272,841	51,731	50,032	47,524	2,073	435
S3	Gray	29.6	31.9	1.85	101.5	1,321,867	658,263	144,086	39,083	37,819	36,236	1,192	391
S4	Mature	51.2	63.4	1.75	91.0	1,537,758	847,293	451,243	126,477	111,186	107,654	2,605	927

Table 3.

Distribution of total OTU’s according to their known, candidatus, and unknown status on each taxonomical level

	Bacteria			Archea
	Known	Candidatus	Unknown	Known	Candidatus	Unknown
Embryo dune (54,500 OTUs)
Phylum	38,725	257	12,105	3278	0	135
Class	32,999	10	18,078	3236	0	177
Order	31,665	10	19,412	3224	0	189
Family	30,187	10	20,890	3210	0	203
Genus	29,875	1191	20,021	2128	21	1264
Species	12,778	860	37,449	502	141	2770
Fore dune (50,032 OTUs)
Phylum	36,018	199	11,742	1856	0	217
Class	33,157	19	14,783	2007	0	66
Order	31,810	19	16,130	1840	0	233
Family	30,094	17	17,848	1836	0	237
Genus	13,757	4028	30,174	1360	0	713
Species	13,937	3548	30,474	1101	111	861
Gray dune (36,236 OTUs)
Phylum	27,686	163	8778	1022	0	170
Class	24,644	10	11,973	1004	0	188
Order	22,617	33	12,977	1004	0	188
Family	22,348	18	14,261	1002	0	190
Genus	19,229	3381	14,017	734	7	451
Species	10,572	3037	23,018	494	60	638
Mature dune (111,186 OTUs)
Phylum	89,561	884	18,136	2266	0	339
Class	84,759	47	23,775	2204	0	401
Order	81,187	47	27,347	2191	0	414
Family	78,109	45	30,427	2190	0	415
Genus	69,379	5704	33,498	1650	35	920
Species	32,660	4469	71,452	1316	174	1115

Relative abundance

Metagenomic study of the Embryo dune sample revealed 27 bacterial phyla comprising the dominant phyla of Actinobacteria (21.8%), followed by Proteobacteria (14.4%), Chloroflexi (11.3%), and Firmicutes (11.3%) (Fig. 1). A total of 12,240 numbers of OTUs were not assigned to any of the phyla. At the class level, the predominance of Actinobacteria (14.66%), followed by Bacilli (10.66%), Alphaproteobacteria (6%), Gammaproteobacteria (4.54%), and Thermoplasmata (4.19%) were recorded (Fig. 2a). Overall, 33.49% of the OTUs were not classified at the class level. At the order level, Bacillales (10.55%) was predominant, followed by Streptomycetales (6.57%) and Methanomassiliicoccales (4.18%) (Fig. 3a). Further, at the family level, Bacillaceae (8.7%), Streptomycetaceae (6.56%), and Methanomassiliicoccaceae (4.15%) were dominant (Fig. 4a). The Embryo dune sample contained the DNA of precisely 339 known genera, dominated by Bacillus (8%), Streptomyces (6.4%), Kouleothrix (6.4%), Methanomassiliicoccus (2.8%), and Acidobacterium (2.2%) (Fig. 5a). Further, 39% of OTUs remained unknown at the Genus level.

Fig. 1.

Major phyla obtained during NGS of Embryo (S1), Fore (S2), Gray (S3), and Mature (S4) dunes samples of Keri beach, Goa-India [major phyla includes 1 and above percent of OTUs]

Fig. 2.

Major classes obtained during NGS of a Embryo (S1), b Fore (S2), c Gray (S3) and d Mature (S4) dunes samples of Keri beach, Goa-India [major classes includes 1 and above percent of OTUs]

Fig. 3.

Major orders obtained during NGS of a Embryo (S1), b Fore (S2), c Gray (S3) and d Mature (S4) dunes samples of Keri beach, Goa-India [major order includes 1 and above percent of OTUs]

Fig. 4.

Major family obtained during NGS of a Embryo (S1), b Fore (S2), c Gray (S3) and d Mature (S4) dunes samples of Keri beach, Goa-India [major family includes 1 and above percent of OTUs]

Fig. 5.

Major genera obtained during NGS of a Embryo (S1), b Fore (S2), c Gray (S3) and d Mature (S4) dunes samples of Keri beach, Goa-India [major genera includes 1 and above percent of OTUs]

Sample from Fore dune showed 22 bacterial phyla and 342 genera. The dominant phylum in the Fore dune was Actinobacteria (19.3%), followed by Proteobacteria (16.3%), Acidobacteria (9.5%), Firmicutes (8.6%), and Verrucomicrobia (5.4%) (Fig. 1). At the class level, Actinobacteria was dominant exhibiting an 11.42% proportion which was followed by Alphaproteobacteria (10.1%), Bacilli (7.94%), and Acidobacteriia (4.62%) (Fig. 2b). At the order level, Bacillales (7.87%) was found to be dominant followed by Rhizobiales (5.71%), Acidobacteriales (4.6%), Chthoniobacterales (3.82%), and Solibacterales (3.02%) (Fig. 3b). Further at the family level, Bacillaceae (5.5%) was dominant, followed by Acidobacteriaceae (4.57%), Chthoniobacteraceae (3.82%), and Solibacteraceae (2.86%) (Fig. 4b). The dominant genus found at the Fore dune was Bacillus (5.2%), followed by “Candidatus Solibacter” (5.1%), Acidobacterium (2.7%), “Candidatus Udaeobacter” (2.5%), Methanomassiliicoccus (2.2%), Gaiella (2.2%), and Streptomyces (2%) (Fig. 5b).

Gray dune comprised of 27 bacterial phyla and 355 genera. Predominant phyla in Gray dune includes Proteobacterium (15.9%), Actinobacteria (15.5%), Acidobacteria (10.6%), Firmicutes (8.2%), Chloroflexi (7.2%), and Verrucomicrobia (6.6%) (Fig. 1). At the class level, Alphaproteobacteria (8.95%) showed dominance, closely followed by Actinobacteria (8.09%), Bacilli (7.16%), Acidobacteriia (5.45%), and Spartobacteria (4.97%) (Fig. 2c). At the order level, Bacillales (7.02%) was the dominant order, followed by Acidobacteriales (5.43%), Chthoniobacterales (4.87%), and Rhizobiales (4.63%) (Fig. 3c). Further at the family level, dominance of Acidobacteriaceae (5.4%), Chthoniobacteraceae (4.87), and Bacillaceae (4.03%) was seen (Fig. 4c). Major genera of bacteria in Gray dune were Kouleothrix (3.9%), Bacillus (3.7%), “Candidatus Udaeobacter” (3.2%), Acidobacterium (3.1%), “Candidatus Solibacter” (2.7%), Gaiella (2.4%), Gemmata (2.1%), and Streptomyces (2.1%) (Fig. 5c).

Mature dune showed the highest bacterial abundance amongst all the dunes. Metagenomics revealed 21 different bacterial phyla and 351 genera. Major phyla included Actinobacteria (26.8%), Proteobacteria (17.6%), Acidobacteria (9%), Firmicutes (8.5%), Bacteroidetes (5.6%), Verrucomicrobia (4.9%), and Planctomycetes (3.1%) in the Mature dune (Fig. 1). At the class level, Actinobacteria was found to be predominant with 21.45% of the total proportion which was followed by Alphaproteobacteria (10.24%), Acidobacteriia (5.06%), and Bacilli (3.79%) (Fig. 2d). At the order level, dominance was shown by Streptomycetales (11.67%), Bacillales (7.85%), Acidobacteriales (5.06%), and Rhizobiales (4.8%) (Fig. 3d). Further at the family level, Streptomycetaceae (11.66%) was found in highest proportion, followed by Bacillaceae (5.62%), Acidobacteriaceae (5.04%), Chitinophagaceae (3.82%), and Chthoniobacteraceae (3.71%) (Fig. 4d). The dominant genera in Mature dune were Streptomyces (11.5%), Bacillus (5.3%), Acidobacterium (4.2%), “Candidatus Udaeobacter” (2%), Sphingomonas (1.8%), and Solirubrobacter (1.5%) (Fig. 5d).

Except the Gray dune, all other dunes have the dominance of Phylum Actinobacteria followed by Proteobacteria. In the Gray dune, Proteobacteria was the dominating phylum, closely followed by Actinobacteria. Genera Aciditerrimonas (Bacteria) and Nitrososphaera (Archaea) were found to be among the major genera in the Embryo dune but not in other dunes. Acidobacterium, Bacillus, Methanomassiliicoccus, Solirubrobacter, and Streptomyces were among the major genera in all the four dunes (Fig. 5). Interestingly, Embryo dune did not show the presence of members from “Candidatus” as its major group of genera. “Candidatus Koribacter”, “Candidatus Solibacter”, and “Candidatus Udaeobacter” were seen as major genera from Fore, Gray, and Mature dunes. Fore and Gray dunes had a similar group of major genera except for Mycobacterium in Fore dune. Based on major genera from all four dunes, Embryo dune had markedly different bacterial and archaeal community structures than Fore, Gray, and Mature dunes. Also, Fore and Gray dunes had a relatively similar community structure than Embryo and Mature dunes. The major species observed from four dunes has been provided in Fig. 6. The total bacterial and archaeal diversity study of CSD from all four dunes using culture-independent technique revealed predominance of genus Streptomyces followed by Bacillus, Acidobacterium, and Kouleothrix (Fig. 7).

Fig. 6.

Major species obtained during NGS of a Embryo (S1), b Fore (S2), c Gray (S3) and d Mature (S4) dunes samples of Keri beach, Goa-India [major species includes 1 and above percent of OTUs]

Fig. 7.

Major genera obtained from CSD using a culture-independent method [major genera includes 1 and above percent of OTUs]

Diversity analysis (alpha diversity, species richness, evenness, dominance, and rarefaction curve)

The Simpson’s and Shannon's alpha diversity indices ranged from 0.9521–0.9764 and 4.143–4.489, respectively. Simpson’s and Shannon's diversity indices were highest in Fore dune followed by Gray, Mature, and Embryo dunes (Table 4). Margalef’s species richness was observed to be highest in Gray dune and lowest in Mature dune (Table 3). Margalef’s species richness of Embryo and Fore dunes is similar and slightly higher than the Mature dune. Evenness or equitability analysis among dunes indicated that the bacterial and archaeal communities were almost evenly distributed in Fore dune and least in Embryo dune (Table 4). Species dominance was highest in Embryo dune and lowest in Fore dune (Table 4). Species dominance of Mature dune is similar to that of Embryo dune, whereas Gray dune’s species dominance is similar to that of Fore dune. The rarefaction curve for the Fore (S2), Gray (S3), and Mature (S4) dune samples in Fig. 8a, b reached a plateau suggesting a good representation of the microbial community as these samples showed a representation of most abundant species with some rare species as well.

Table 4.

Genera level diversity indices calculated from Next Generation Sequencing approach

Diversity indices	Embryo (S1)	Fore (S2)	Gray (S3)	Mature (S4)
Total taxa	339	342	355	351
Total OTU’s	33,201	30,887	23,351	76,768
Dominance (D)	0.0478	0.0236	0.0254	0.0448
Simpson#	0.9521	0.9764	0.9746	0.9551
Shannon (H’)	4.143	4.489	4.412	4.274
Pielou’s evenness (J’)	0.1857	0.2603	0.2323	0.2046
Margalef’s richness (d)	32.47	32.98	35.19	31.12

(D) = sum [(n_i/n)²] where n_i is the number of individuals of taxon _i, (#) = 1-dominance, (H’) = -sum [(n_i/n) log n(n_i/n)], (d) = [(S − 1)/log n(n)], (J’) = (H’/H’_max), (S) = a*log n(1 + n/a), where S is number of taxa, n is number of individuals and H’_max is maximum possible value of H’

Fig. 8.

a Rarefaction curve: observed species and b Rarefaction curve: Chao1 curves obtained from all the four sub-dune samples namely; Embryo (S1), Fore (S2), Gray (S3), and Mature (S4) dunes

Beta diversity among dunes

Beta diversity (Whittaker’s) was calculated based on ‘Bray–Curtis dissimilarity’ at the species level. It calculates the differences in species abundance among multiple samples. Beta diversity range from 0 to 1 (0 refers that two samples have the same species at the same abundance; whereas one completely different species abundance in two samples). The highest beta diversity was observed between Gray and Embryo (0.38), followed by Mature and Embryo (0.37), then Fore and Embryo (0.35), and Mature and Gray dunes (0.25); whereas, the lowest beta diversity was observed between Gray and Fore (0.19) and Mature and Fore dunes (0.19) (Table 5).

Table 5.

Pairwise comparison between Embryo (S1), Fore (S2), Gray (S3), and Mature (S4) dunes for determining beta diversity based on ‘Bray–Curtis dissimilarity’ at species level

Dunes	Embryo (S1)	Fore (S2)	Gray (S3)	Mature (S4)
Embryo (S1)	0	0.35683	0.3804	0.37101
Fore (S2)	0.35683	0	0.19369	0.19769
Gray (S3)	0.3804	0.19369	0	0.25779
Mature (S4)	0.37101	0.19769	0.25779	0

Extremophiles in the dunes

Percent distribution of alkaliphiles, acidophiles, thermophiles, methanotrophs, and radiotolerant within each dune is presented in Table 6. Acidophiles, thermophiles, methanogens, and radiotolerants were present in all the four dunes. Families containing alkaliphiles were found in three dunes except Mature dune. Among the three dunes, percent contributions of alkaliphiles were found highest in Embryo dune. Among extremophiles, maximum proportion of acidophiles were seen in Fore, Gray, and Mature dunes. Within the dunes, the highest proportions of acidophiles were found in Gray dune followed by Mature, Fore and Embryo dune. Halophiles were present only in Embryo and Gray dune. Among extremophiles, maximum numbers of diverse families were observed for thermophiles. Within the dunes, highest proportion of thermophiles was found in Fore and Mature dunes followed by Gray and Embryo dunes. Among extremophiles, maximum proportion of methanotrophs was seen in Embryo dune followed by Fore, Gray, and Mature dunes. Within the dunes highest proportion of radiotolerants were found in Embryo dune followed by Gray, Fore, and Mature dunes.

Table 6.

Distribution of extremophiles in the coastal sand dunes

Extremophiles	Families	Dunes
		Embryo (%)	Fore (%)	Gray (%)	Mature (%)
Alkaliphiles	Alcanivoracaceae	0.32	ND	0.01	ND
	Egibaceraceae	0.03	0.03	ND	ND
	Egicoccaceae	0.12	ND	ND	ND
Total (%)		0.47	0.03	0.01	ND
Acidophiles	Acetobacteraceae	0.22	0.44	0.52	0.24
	Acidiferrobacteraceae	ND	ND	ND	0.01
	Acidimicrobiaceae	1.38	1.07	1.25	0.73
	Acidobacteriaceae	2.39	4.57	5.4	5.04
	Acidothermaceae	ND	0.02	ND	ND
Total (%)		3.99	6.1	7.17	6.01
Halophiles	Halomonadaceae	0.67	ND	ND	ND
	Alcanivoracaceae	0.32	ND	0.01	ND
	Egicoccaceae	0.12	ND	ND	ND
	Saccharospirillaceae	0.02	ND	ND	ND
Total (%)		1.13	ND	0.01	ND
Thermophiles	Desulfovibrionaceae	0.06	0.06	0.04	0.02
	Hydrogenophilaceae	0.01	ND	0.01	ND
	Sinobacteraceae	0.27	0.34	0.17	0.5
	Sphaerobacteraceae	0.23	0.29	0.31	0.24
	Tepidisphaeraceae	0.36	1.42	1.12	1.4
	Thermoactinomycetaceae	0.09	0.04	0.09	0.05
	Thermaceae	ND	ND	0.01	ND
	Thermoanaerobacteraceae	0.07	0.07	0.11	0.05
	Thermoanaerobacterales Family III. Incertae sedis	0.01	0.01	0.01	0.01
	Thermodesulfobacteriaceae	0.01	0.01	0.01	0.01
	Thermoflexaceae	0.02	0.01	0.013	ND
	Thermogemmatisporaceae	0.01	ND	0.01	ND
	Thermoleophilaceae	0.25	0.20	0.13	0.19
	Thermolithobacteraceae				0.01
	Thermomicrobiaceae	0.04	0.04	0.04	0.04
	Thermomonosporaceae	0.08	0.29	0.3	0.29
	Thermotogaceae	0.04	ND	ND	ND
Total (%)		1.6	2.83	2.43	2.84
Methanotrophs	Methanomassiliicoccaceae	4.15	2.37	1.77	1.2
	Methylobacteriaceae	0.5	0.32	0.28	0.41
	Methylococcaceae	0.01	0.01	0.01	0.01
	Methylocystaceae	0.03	0.02	0.07	0.04
	Methylophilaceae	0.03	ND	ND	0.01
Total (%)		4.75	2.72	2.13	1.67
Radiotolerant	Rubrobacteraceae	0.51	0.15	0.17	0.08
	Deinococcaceae	0.04	ND	ND	ND
Total (%)		0.55	0.15	0.17	0.08

ND not detected

Exclusive and common members among dunes

Venn diagrams showing relationships among the four dunes were constructed to learn the common and exclusive members present at phyla (Fig. 9), classes (Fig. 10), genera (Fig. 11a), and species (Fig. 11b) levels. Among the identified phyla, 29, 24, 29, and 23 phyla were found in Embryo, Fore, Gray, and Mature dunes, respectively. Four phyla viz. Fibrobacteres, Thermodesulfobacteria, Thermotogae, and Balneolaeota were exclusively found in the Embryo dune (Fig. 9), and constituted about 0.32% of the total OTUs. Three phyla namely, Nitrospinae, Candidate division NC10, and Spirochaetes were exclusively found in Gray dune which constituted 0.042% of the total OTUs. Sixty-three percent of total phyla identified from CSD were present across all dunes (Fig. 9). The proportion of commonly found phyla for each dune ranged from 72 to 91%.

Fig. 9.

Venn diagram exhibiting the exclusive and common phyla in a culture-independent analysis of Embryo (S1), Fore (S2), Gray (S3) and Mature (S4) dunes of Keri beach, Goa-India

Fig. 10.

Venn diagram exhibiting the exclusive and common at class level in a culture-independent analysis of Embryo (S1), Fore (S2), Gray (S3) and Mature (S4) dunes of Keri beach, Goa-India

Fig. 11.

Venn diagram exhibiting the exclusive and common a genera and b species in a culture-independent analysis of Embryo (S1), Fore (S2), Gray (S3) and Mature (S4) dunes of Keri beach, Goa-India (Listing in supplementary Table 1 and 2)

The numbers of classes in Embryo, Fore, Gray, and Mature dunes were 57, 50, 53, and 52, respectively. About 74% of classes identified from CSD were present across all the four dunes (Fig. 10). The five exclusive classes found in Embryo dune namely Thermodesulfobacteria, Methanomicrobia, Thermotogae, Balneolia, and Fibrobacteria, constituted about 0.33% of the total OTUs. The exclusive class found in Gray dune is Nitrospinia, which constituted about 0.013% of the total OTUs. Exclusive classes found in Mature dunes are Oligoflexia, Thermolithobacteria, and Methanococci, which constituted about 0.042% of total OTUs. The proportion of commonly found classes for each dune ranged from 80 to 92%. About 92% of classes found in Mature dune are present in Gray dune and 90% of classes from Gray dune exist in Mature dune. From Embryo dune, 86% of classes are found in Fore dune and 96% of classes from Fore dune exist in Embryo dune. From Fore dune, 98% of classes are found in Gray dune and from Gray dune, 92% of classes are present in Fore dune.

The numbers of genera found in Embryo, Fore, Gray, and Mature dune are 339, 342, 355, and 351, respectively. Listing of common and exclusive genera is provided in Online Resource 2. About 35% of genera identified from CSD were present across all the four dunes (Fig. 11a). The proportion of commonly found genera for each dune ranged from 54 to 57% (Fig. 11a). About 75% of the genera are found common among Mature and Gray dunes. Similarly, 65% of genera are found common among Embryo and Fore dunes. Further, 82% of genera are found common in Fore and Gray dunes. Embryo dune has the highest number of exclusively found genera (25%) followed by Mature and Gray dunes (12% both) and lastly Fore dune (5%).

Embryo, Fore, Gray, and Mature dunes consisted of 445, 437, 435, and 520 species, respectively. Listing of common and exclusive species is provided in Online Resource 3. About 16% of total species identified from CSD were found across all the four dunes (Fig. 11b). The proportion of commonly found species for each dune ranged from 30 to 35% (Fig. 11b). About 52% of species from Mature dune are shared by Gray dune and 63% species of Gray dune were shared by Mature dune. About 45% of species identified from Embryo dune exist in Fore dune and 46% of species from Fore dune are found in Embryo dune. About 65% of species found common in Fore and Gray dunes.

Heatmap analysis

Fore and Gray dunes showed clustering together in a heatmap at genera level indicating the similarity in members found. Mature dune formed an outgroup indicating the presence of distinct members from that of other dunes (Fig. 12). Mature dune has more prominent heat signatures as compared to other dunes. Fore dune showed the least heat signatures as compared to other dunes. It is clear from the heatmap that the members showing low counts in Mature dune showed higher heat signatures in the other dunes and vice versa. The heatmap of genera level also showed a similar pattern to that of the phylum level (Fig. 13).

Fig. 12.

Heatmap generated using the relative abundance of bacteria at the phyla level from metagenomics analysis. The X-axis represents the four dunes of CSD and Y-axis represents the phyla level taxon

Fig. 13.

Heatmap generated using the relative abundance of bacteria at the genera level from metagenomics analysis. The X-axis represents the four dunes of CSD and Y-axis represents the genera level taxon

UPGMA cluster tree analysis

A UPGMA tree was constructed from raw consensus sequences based on the unweighted uniFrac approach of the metagenomics analysis of Embryo, Fore, Gray, and Mature dune samples. This method is based on sequence distances, the fraction of branch length which is shared between samples, and the uniqueness of the sample. Fore and Gray dunes were clustered together indicating similarity in members found between them (Fig. 14). Embryo and Mature dunes formed a distant outgroups indicating a uniqueness of sequences in the sample.

Fig. 14.

UPGMA cluster tree drawn using consensus sequences of Embryo (S1), Fore (S2), Gray (S3), and Mature (S4) dunes based on unweighted uniFrac approach

Discussion

CSD acts as a crucial and delicate barrier between the high saline oceans and the terrestrial habitat. CSD ecosystem is dynamic and acts as a unique habitat for life forms. Being dynamic, changes occurring in this ecosystem pose continuous challenges to life forms that include sparse nutrition and extreme physio-chemical conditions. The dune as described earlier is divided into various dunes, each of which presents a large number of challenges to the life forms. It is envisaged, therefore, that the life forms present in these will have diverse capabilities to withstand the dynamic and stressed conditions. However, the understanding of microbial biodiversity for this specialized ecosystem is negligible.

In recent years, few attempts have been made to study microorganisms from CSD and these have been demonstrated to have various ecological roles (Shinde et al. 2020; Nayak et al. 2013; Muthukumar and Samuel 2011; Aureen et al. 2010; Gaonkar et al. 2012). For example, some of the bacteria from CSD such as Pseudomonas, Bacillus and Microbacterium are capable of producing exopolysaccharide in large quantities that get associated with sand particles and forms aggregates, thereby providing stability to the unstable dune (Nayak et al. 2019; Godinho and Bhosle 2009). Also, CSD harbors various bacteria that have potential in plant growth promotion (Aureen et al. 2010; Muthukumar and Samuel 2011), bioremediation (Gaonkar and Bhosle 2013), and hydrocarbon and tarball degradation (Gaonkar et al. 2012; Shinde et al. 2020).

The present study focuses on the bacterial and archaeal diversity in the four zones of CSD namely Embryo, Fore, Gray, and Mature dunes using a metagenomics approach. This study is the first detailed characterization of CSD microbiota in four dunes. This study attempts to understand the bacterial and archaeal diversity, abundance, different phyla, genera, and species present in these four dunes. NGS technology helps in exploring the microbial diversity and community structure of any given environmental sample (water, soil, sediment, sand). The use of Illumina (HiSeq) sequencing of the V3–V4 hypervariable regions of the 16S rRNA gene, gave an idea of bacterial and archaeal communities residing in different dunes of CSD. From NGS of samples from Embryo, Fore, Gray, and Mature dunes, the highest numbers of filtered OTUs were obtained from Mature dune whereas, the lowest was obtained from Gray dune. Thus, indicating that Mature dune had a higher abundance and Gray dune had the lowest abundance of bacteria and archaea. Mature dune has high humus content and vegetation as compared to other dunes which could facilitate the growth of microorganisms (McLachlan and Brown 2006). A sample of Mature dune was collected from the rhizosphere. Root exudes of plants in the rhizosphere makes it a unique niche that enables the growth of several microorganisms (Glick 2012). This could be another reason for higher abundance in Mature dune.

Archaeal phyla namely Euryarchaeota and Thaumarchaeota were present across all four dunes. Members of Thaumarchaeota are known to tolerate extreme environmental conditions and perform the nitrification process whenever ammonia concentration in soil is low (Zhang and He 2012; Sauder et al. 2011). Embryo and Gray dune samples showed the presence of 27 bacterial phyla whereas, Fore and Mature dunes showed 22 and 21 phyla, respectively. Phylum Actinobacteria was found to be predominant in Embryo, Fore, and Mature dunes, followed by Proteobacteria. However, Gray dune showed a dominance of phylum Proteobacteria, closely followed by Actinobacteria. Lin et al. (2014) also reported the predominance of phyla Actinobacteria and Proteobacteria from CSD using a culture-independent study. A culture-dependent study on the diversity of Sand dunes of Qatari Barchan showed a predominance of Actinobacteria (Abdul et al. 2016). The total GC content of the samples ranged from 53 to 56%, indicating the higher possibilities of finding the majority of the genera belonging to Actinobacteria. Members of Actinobacteria are known to have high GC content (Verma et al. 2013). Actinobacteria are ubiquitously found taking part in various environmental processes including nitrogen fixation, decomposition, and various nutrient cycles (Mohammadipanah and Wink 2016). Studies on microbial diversity of marine and terrestrial ecosystems such as paddy field soil (Li et al. 2019), rice straw compost (Wang et al. 2016), and oxygen minimum zone of Bay of Bengal (Fernandes et al. 2019), have shown Actinobacteria as dominant phyla. Bolhuis and Stal (2011) reported the dominance of phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria in the coastal microbial mats of the North Sea beaches of the Dutch barrier island Schiermonnikoog. Abdul et al. (2016) recorded the predominance of the genus Enterobacteria. The current study showed Bacillus as a dominant genus in the Embryo and Fore dunes, whereas Kouleothrix and Streptomyces were dominant genera in Gray and Mature dunes, respectively. Scientists have tried to estimate the bacterial diversity in the rhizosphere of CSD vegetation. Shin et al. (2007) reported the predominance of Pseudomonas fluorescens in the rhizosphere of Sphagnum and Tortula present in CSD of the Tae-An area. Lee et al. (2006) reported the predominance of Lysobacter in the rhizosphere of Calystegia soldanella and Elymus mollis of CSD of Tae-An, Chungnam Province, Korea, using 16S rRNA gene clones. Park et al. (2005) reported the dominance of Pseudomonas, Chryseobacterium, and Arthrobacter in the rhizosphere of CSD plants in a culture-dependent study and stated that the bacterial diversity at CSD is high and unique.

Further, the present study reported members of Chloroflexi phylum contributing a major proportion in all the dunes (about 7% of total OTUs). They are known as ‘metabolic specialists’. They are anaerobic phototrophs, thermophiles, and halorespirers (Speirs et al. 2019). Hence, it is difficult to obtain them using culture-based methods. Therefore, the metagenomics approach was useful in assessing the microbiota, which in culture-based methods would have been missed.

Observed species richness is dependent on the size of the sample collected. It is almost impossible to measure actual species richness which is a natural measure of biodiversity (May 1988). Several statistical methods can approximately estimate the species richness, diversity indices, which include Margalef’s species richness (d), Shannon’s diversity index, Fisher’s alpha, Chao1, rarefaction curve, and more. Evenness and abundance in a sample are the two main factors that influence biodiversity. Abundance is the number of individuals per species and evenness is how equally abundant species are on a site. All the species are hardly equally abundant in a particular habitat simply because some of the species are better competitors than the others. Evenness is considered a good indicator whereas the dominance of single species is considered a disturbance in an ecosystem.

An attempt is made to understand biodiversity in terms of above multivarient characteristics. From the present study, it is established that the diversity indices of CSD ranged from 4.1 to 4.4. Lee et al. (2006) report the diversity indices (Shannon–Weaver) of the rhizosphere of CSD plant to be 4.91 and 4.02 of Calystegia soldanella and Elymus mollis, respectively. The present study established that Fore dune has the highest bacterial and archaeal diversity and lowest species dominance compared to other dunes. Embryo dune showed the highest dominance which implied the least diversity compared to other dunes which were also confirmed by Shannon and Simpson’s alpha diversity indices. Also, bacterial and archaeal communities at Embryo dune were most evenly distributed; this was calculated using Pielou’s evenness (J’). The Embryo dune is a very fragile habitat for microbial fauna due to the constant tidal effect. Hence, the dune is not diverse and rich in species.

Thermophiles, acidophiles, methanotrophs and radiotolerant were found in all the four dunes in the present study. Alkaliphiles were present in Embryo, Fore, and Gray dunes, whereas halophiles were found in Embryo and Gray dunes. Alkaliphiles and halophiles are known to be present in the Embryo and Fore dunes. Since, the salinity and pH is higher due to sea water and calcification of sea shells in these dunes (Godinho and Bhosle 2009). Additionally, their proportion is also higher in Embryo dune, since it is closest to and most influence from sea. Acidophiles are present in major proportion in the Gray followed by Fore and Mature dunes. Also, Embryo dune has the least proportion. The presence of humus contributes to the acidity of the soil. Gray, Mature, and Fore dunes have substantial amount of vegetation in the Keri beach, Goa, India, thus leading to enrichment of sand with humus. Members of family Rubrobacteraceae has high tolerance towards extremely ionizing radiations and are also thermophilic in nature (Rosenberg et al. 2014). Members of this family were primarily isolated from hot springs. From current study, they were obtained from all the four dunes. Since the entire CSD ecosystem receives high level of UV radiations. Additionally, Embryo dune having no vegetation and more exposed to UV rays was found to have major proportion of radiotolerant microbes.

Although 156 species and 193 genera of bacteria and archaea were commonly found in all the dunes, there were exclusively members in each of the dunes. Both at genera and species level, a similar pattern of exclusively found members observed, viz., Embryo dune showed the highest number of exclusively found genera followed by Mature, Gray, and lastly Fore dunes. Mature dune comprised 6% higher exclusive species than Gray dune, unlike in genera level. Embryo dune has the highest number of the exclusive members at all the taxonomical levels, i.e., phyla (4), class (5), genera (85), and species (195) than any other dunes. These exclusive taxons are not found in the other dunes of CSD. Bacillus deserti was found exclusively in the Embryo dune and no other dunes. This species of Bacillus has been previously reported from desert soil of Xinjiang Province, China (Zhang et al. 2011). Additionally, Domibacillus indicus was found exclusively in Embryo dune, which was previously isolated from the marine sediment of Lakshadweep, India (Sharma et al. 2014). Marinobacter bryozoorum found exclusively in Embryo dune is a marine and halophilic bacterium. Therefore, Embryo dune is a unique niche that has bacteria and archaea markedly present in harsh environments. Spongiibacter sp., a halophile usually present in association with marine sponges such as Haliclona sp. (Graeber et al. 2008), was found to be present exclusively in Embryo dune during this study. There were several genera exclusive to Embryo dune which were halophiles and specifically found in marine eco-niches. These genera are Oceanococcus, Marinobacter, Oceanibacterium, Marinoscillum, Spongiibacter, and Halophibacterium. No members of these genera were present in other dunes.

Fore dune of Keri, Goa has the least amount of exclusive genera and species. Bacillus thuringiensis is found exclusively in the Fore dune. Almeida et al. (2020) has reported B. thuringiensis from the coast of Miramar, Goa, India, possessing mosquito pathogenicity. Rhizobium gallicum and R. etli, exclusive to Fore dune are root-nodule bacteria capable of fixing atmospheric N₂. These species are known to be associated with legumes such as Phaseolus vulgaris and Vigna unguiculata, which could be used for plant growth promotion (Mhamdi et al. 2002).

Gray dune has 101 exclusive bacterial and archaeal species that are not present in any other dunes. Rhodovibrio salinarum, exclusive to Gray dune is a halophilic bacterium and usually found in salterns (Imhoff 2015). Also, various beneficial species were found exclusive to the Gray dune such as hexane-degrading Alkanibacter difficilis, Nitrogen-fixing Paraburkholderia sabiae, Polyphosphate-accumulating Microlunatus phosphovorus and cellulase, amylase, and polygalacturonate lyase-producing Thermomonospora curvata. Therefore, these microorganisms from Gray dune have tremendous potential in various fields. Gray dune showed the presence of Aquabacterium limnoticum and Hydrobacter penzbergensis, that had been reported earlier from freshwaters (Chen et al. 2012; Eder et al. 2015), indicating the ability of these species to survive in two completely different environments.

Mature dune has 154 exclusive bacterial and archaeal species that are not found in other dunes. Sorangium cellulosum is an exclusively found bacterial species in Mature dune. It is a saprophyte and mostly gains nutrition from cellulose-containing organic compounds. It is reported to have fungicidal and bactericidal capabilities (Pradella et al. 2002). Thus, this species could have a role in plant growth promotion in the CSD environment. The Genus Vulcanibacillus is exclusively present in Mature dune and was previously reported from hydrothermal vents in the rainbow vent field (l' Haridon et al. 2006). So far, only one species has been identified from this genus which is recorded in the current study, i.e., V. modesticaldus. It is anaerobic, moderately thermophilic, and capable of reducing nitrate. Brevibacillus brevis, exclusively found in the Mature dune, is a known producer of antibiotics-gramicidin, tyrocidine, and is also a fungicide (Chandel et al. 2010). Also, few strains of B. brevis are capable of oxidizing carbon monoxide.

The present study identified several pigment-producing species from CSD such as Spirosoma aerolatum (yellow), Micromonospora auratinigra (deep orange), Acidisphaera (salmon-pink), Chromohalobacter nigrandesensis (black), Domibacillus indicus (red), Micromonospora narathiwatensis (pale yellow), Pontibacter saemangeumensis (pink), Pseudomonas fluorescens (Pyoverdine) and Rhodococcus equi (red). Several culture-dependent studies have shown the presence of pigment-producing bacteria in CSD habitat. This could be because pigmentation by bacteria and archaea is a mechanism to combat stress conditions such as salt stress, high UV rays, and metal contamination (Narsing et al. 2017). CSD environmental condition is harsh and extreme and the presence of pigmented bacteria in CSD is inevitable.

This study is the first metagenomics approach in assessing the microbiota in transactional zones of tropical CSDs. Data from the current work clearly demonstrate the dynamic nature of microbial communities of CSD at four dunes. The present work establishes the distinctiveness of each dune in terms of their microbial community structure. Moreover, using the metagenomics approach to assess microbial diversity in the CSD provides a better assessment of potential bacteria and archaea along with its community structure. Furthermore, a study of gene expression with CSD isolates would give a better idea of the adaptive mechanisms microorganisms use to sustain themselves in harsh conditions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the study conception and design. Materials preparation, data collection, and analysis were performed by SAS and SG. The draft of the manuscript was written by SAS and SG commented on previous versions of the manuscript. All the authors read and approved the final manuscript.

Funding

This work was supported by the University Grant Commission (UGC), National Fellowship for Students to pursue a Ph.D. degree [award letter number: F./2014-15/NFO-2014-15-OBC-GOA-486/(SA-III/website)].

Availability of data and materials

GenBank submission All the Next-generation sequences have been submitted to GenBank and have appeared in the public database.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.