3.4 Taxonomic analysis of bacterial community abundance on Roccella montagnei
The bacterial community abundance was analyzed in Roccella montagnei for all taxonomic levels and the results were observed as follows, their respective stacked bar plots.
The most abundant phyla in the 3 lichen Roccella montagnei samples, which were in coastal and terrestrial habitat were (i) Proteobacteria with 95.78% present in Nagercoil lichen sample, 96.54% in Pollachi lichen sample, and a 96.16% in lichen sample collected from Vedaranyam; (ii) Bacteroidetes having a presence of 1.36% in Nagercoil lichen sample, 1.87% in Pollachi lichen sample and 1.77% in Vedaranyam sample. The other phyla are in decreasing order as follow: Actinobacteria, Acidobacteria, Firmicutes, Unclassified phyla, Cyanobacteria, Fusobacteria, Gemmatimonadetes, Tenericutes, WPS-2, Chloroflexi, Verrucomicrobia (Fig. 4 (a)). There are 4 common and significantly abundant classes in all 3 corticoles lichen Roccella montagnei grown under coastal and terrestrial habitat in the tree bark of Cocos nucifera and Maba buxifolia. (i) Gammaproteobacteria: 65.68% in Nagercoil lichen in Cocos nucifera (Coastal), 58.92% in Pollachi lichen from Cocos nucifera (Terrestrial), 56.85% in Vedaranyam lichen from Maba buxifolia (Coastal). (ii) Alphaproteobacteria: 25.71% in Nagercoil sample, 32.43% in Pollachi sample, 34.73% in Vedaranyam sample; (iii) Betaproteobacteria: 4.36% in Nagercoil sample, 4.96% in Pollachi sample, 4.53% in Vedaranyam sample; (iv) Flavobacteriia: 1.14% in Nagercoil sample, 1.81% in Pollachi sample, 1.55% in Vedaranyam sample. The other classes are in decreasing order as follows: Actinobacteria, Acidobacteriia, Unclassified classes, Bacilli, Saprospirae, Deltaproteobacteria, Chloroplast, Clostridia, Oscillatoriophycideae, Fusobacteriia, Gemmatimonadetes, Mollicutes, Solibacteres, Synechococcophycideae, Rubrobacteria, Anaerolineae, Sphingobacteriia, Acidimicrobiia, Spartobacteria (Fig. 4 (b)). There are about 9 significantly abundant and orders common to all the 3 corticolous lichen Roccella montagnei grown under coastal and terrestrial habitat in the tree bark of Cocos nucifera and Maba buxifolia. (i) Pseudomonadales: 42.04% in Nagercoil sample, 34.96% in Pollachi sample, 33.61% in Vedaranyam sample, (ii) Rhizobiales: 15.99% in Nagercoil sample, 27.89% in Pollachi sample, 29.1% in Vedaranyam sample, (iii) Xanthomonadales: 10.98% in Nagercoil sample, 17.48% in Pollachi sample, 8.82% in Vedaranyam sample, (iv) Enterobacteriales: 12.46% in Nagercoil sample, 6.27% in Pollachi sample, 12.85% in Vedaranyam sample, (v) Burkholderiales: 4.3% in Nagercoil sample, 4.9% in Pollachi sample, 4.48% in Vedaranyam sample, (vi) Rhodospirillales: 5.73% in Nagercoil sample, 0.74% in Pollachi sample, 1.77% in Vedaranyam sample, (vii) Caulobacterales: 2.4% in Nagercoil sample, 2.3% in Pollachi sample, 1.74% in Vedaranyam sample, (viii) Sphingomonadales: 1.31% in Nagercoil sample, 1.27% in Pollachi sample, 1.98% in Vedaranyam sample, (ix) Flavobacteriales: 1.14% in Nagercoil sample, 1.81% in Pollachi sample, 1.55% in Vedaranyam sample (Fig. 5 (c)). In family level there are about 11 common important families abundant in the 3 lichen corticolous lichen Roccella montagnei grown under coastal and terrestrial habitat in the tree bark of Cocos nucifera and Maba buxifolia. (i) Pseudomonadaceae: 32.9% in Nagercoil sample, 24.75% in Pollachi sample, 26.21% in Vedaranyam sample, (ii) Brucellaceae: 7.92% in Nagercoil sample, 23.41% in Pollachi sample, 18.14% in Vedaranyam sample, (iii) Xanthomonadaceae: 10.89% in Nagercoil sample, 17.48% in Pollachi sample, 8.67% in Vedaranyam sample, (iv) Enterobacteriaceae: 12.46% in Nagercoil sample, 6.27% in Pollachi sample, 12.85% in Vedaranyam sample, (v) Moraxellaceae: 9.14% in Nagercoil sample, 10.21% in Pollachi sample, 7.4% in Vedaranyam sample, (vi) Alcaligenaceae: 3.41% in Nagercoil sample, 4.4% in Pollachi sample, 3.67% in Vedaranyam sample, (vii) Methylocystaceae: 1.99% in Nagercoil sample, 2.28% in Pollachi sample, 5.98% in Vedaranyam sample, (viii) Rhizobiaceae: 4.03% in Nagercoil sample, 0.91% in Pollachi sample, 3.27% in Vedaranyam sample, (ix) Acetobacteraceae: 5.72% in Nagercoil sample, 0.74% in Pollachi sample, 1.75% in Vedaranyam sample, (x) Caulobacteraceae: 2.4% in Nagercoil sample, 2.3% in Pollachi sample, 1.74% in Vedaranyam sample, (xi) Sphingomonadaceae: 1.31% in Nagercoil sample, 1.27% in Pollachi sample, 1.97% in Vedaranyam sample (Fig. 5 (d)). Eight commonly noteworthy genera are present in all 3 corticolous lichen Roccella montagnei grown under coastal and terrestrial habitat in the tree bark of Cocos nucifera and Maba buxifolia Along the genera level, one genus is signified as unclassified. (i) Unclassified: 32.34% in Nagercoil sample, 13.35% in Pollachi sample, 26.79% in Vedaranyam sample, (ii) Pseudomonas: 23.75% in Nagercoil sample, 24.42% in Pollachi sample, 23.06% in Vedaranyam sample,(iii) Ochrobactrum: 7.84% in Nagercoil sample, 21.27% in Pollachi sample, 18.09% in Vedaranyam sample, (iv) Stenotrophomonas: 10.15% in Nagercoil sample, 17.47% in Pollachi sample, 8.05% in Vedaranyam sample, (v) Acinetobacter: 9.12% in Nagercoil sample, 10.19% in Pollachi sample, 7.4% in Vedaranyam sample, (vi) Achromobacter: 3.39% in Nagercoil sample, 4.37% in Pollachi sample, 3.63% in Vedaranyam sample, (vii) Agrobacterium: 3.83% in Nagercoil sample, 0.79% in Pollachi sample, 3.14% in Vedaranyam sample, (viii) Brevundimonas: 0.7% in Nagercoil sample, 2.05% in Pollachi sample, 0.52% in Vedaranyam sample (Fig. 6 (e)).
3.5 Alpha Diversity analysis of the lichen microbiome communities
Alpha diversity is mainly used to reflect the diversity of each lichen sample, which estimates the number of species in the microbial community as well as the abundance and diversity of species in environmental communities through a series of statistical indices. In which are summarized in terrestrial and coastal habitat lichens in distinct geographic locations of tree as growing substrate of Cocos nucifera and Maba buxifolia in Nagercoil, Vedaranyam, Pollachi of Tamil Nadu, India. The indices for community richness calculation include: Chao and ACE index. The Shannon and Simpson index for community diversity calculations. The Goods coverage indices to measure sequencing depth (Coverage). Nagercoil lichen had the highest Shannon index (4.75) which indicates abundance of the lichen associated bacterial community present in a significantly maximum in the trees of Cocos nucifera in coastal habitat in comparison with other two lichen samples i.e., Pollachi sample (4.12) and Vedaranyam sample (4.515).
The Simpson’s index shows that quantify the diversity of species richness and evenness is almost the same in Nagercoil lichen from Cocos nucifera in terrestrial locations (0.923) and Vedaranyam lichen from Maba buxifolia in Coastal locations (0.909) and both the samples were higher than the Pollachi Cocos nucifera in terrestrial location (0.884).
The Fig. 7 (a) shows the rarefaction curve i.e., it tries to determine all of the bacterial diversity in a sample at a particular sequencing depth by sub sampling the sequence to a minimum and plotting it against the observed OTUs which enables the comparison among the different lichen samples in of different and same tree substrate in distinct geographical locations. The curves represent the bacterial diversity as a function of sequencing depth. The X axis is the number of valid sequences extracted, and the Y axis is the number of OTUs (Observed OTUs). Each sample is represented by one curve with a unique color. The number of OTUs increases with the increase of extracted sequence count until reaching a plateau, which indicates the number of detected OTUs will not increase with the number of extracted sequences and reflects the reasonable sequence depth.
Rank-abundance curve is used to analyze bacterial diversity. Rank-abundance curve reflects both bacterial species abundance and species uniformity in lichen. The abundance of species is reflected by the length of the curve on the X axis. The more extended on the X axis, the more abundant the species is. Species uniformity is reflected by the shape of the curve. The smoother the curves, the higher the species uniformity in lichen Roccella montagnei.
Each curve in the figure above corresponds to an individual lichen Roccella montagnei samples from three locations. The X axis is the relative abundance of the OTU in descending order. The Y axis is the relative abundance of the OTU. '100' on the X axis indicates the OTU in the sample is ranked as the 100th abundant in descending order, and the corresponding value on the Y axis is the percentage of the sequence count in the OTU (the number of sequences of the OTU divided by the total number of sequences) (Fig. 7 (b)).
3.6 Beta diversity of the lichen microbiome communities:
Beta diversity analysis is used to determine how different the lichen bacterial community in coastal samples is from that in terrestrial habitats, as well as to disclose elements of microbial ecology that aren't apparent simply looking at the sample composition. Beta diversity analysis is frequently divided into two categories. They can be quantitative (e.g., Bray-Curtis or weighted UniFrac) or qualitative (e.g., Bray-Curtis or weighted UniFrac) (Goodrich et al. 2014). The NMDS, PCA, and PCoA figures were all created using beta diversity distance matrices.
3.6.1 PCA and PCoA plot analysis:
PCA analysis (Principal Component Analysis): The difference and distance between samples can be reflected by the analysis of the gene functional distribution of different samples of Roccella montagnei. The first, second, and third main components are represented by PC1, PC2, and PC3, respectively (Fig. 8 (a)). The percentage after the main component, which reflects how much information the primary component can extract from the original data, represents the contribution rate of this component to sample difference. The distance between coastal and terrestrial lichen demonstrates the similarity of the distribution of functional categories in the sample.
The PCoA (Principal Co-ordinates Analysis) is a method for analysing and visualising data differences and similarities. Illustrations from the same group are identified by the similar colour and form. The PCoA plot for the first and second principal coordinates is shown in the PC1 vs PC2 image (Fig. 8 (b)), with the first and second axes representing the first and second principal coordinates, respectively. The contribution of the related coordinate to the sample variations is shown by the percentage number in the axis label, which illustrates how much information is recovered from the original data. The distance between the sample points demonstrates the similarity of the bacterial population in the lichen.
According to the principle component analysis (PCA) and Principal Co-ordinates Analysis (PCoA), the lichen samples from Nagercoil, Pollachi, and Vedaranyam were differed significantly. Mostly, the Nagercoil lichen from Cocos nucifera in coastal habitat and Vedaranyam lichen from Maba buxifolia in coastal locations, indicated similarity than the other sample comparisons.
3.6.2 UPGMA Tree
Cluster analysis uses evolutionary data from sample sequencing to determine if samples in a well-known environment are significantly different from an evolutionary branch in bacterial communities. In UPGMA tree each branch represents a sample and difference of colours will represent different groups present. The results (Fig. 9), infers the all the 3-lichen bacterial community belongs to the same group and the evolutionary microbial lineage between Nagercoil lichen from Cocos nucifera from coastal habitat and Vedaranyam lichen from Maba buxifolia from Coastal habitat are similar and the highly different from the Pollachi lichen from Cocos nucifera from terrestrial habitat.