**3. Results**

The minimum distance between any two trees was about 1 m, and the maximum was approximately 30 m. We collected one soil sample of each sampling unit (tree) during the months of the study. Soil samples were sieved in the field with a 2 mm mesh and placed in plastic bags. Samples were then placed on ice, taken to the laboratory, and frozen until they were sent to the Molecular Research Facility at Lubbock Texas. Total soil DNA extraction and 454 pyrosequencing were completed at the Molecular Research Facility. The molecular research facility reported all

Enzyme activities were performed as described in [5, 15]. The activities of

glucosaminidase), P cycling (alkaline phosphatase, acid phosphatase, phosphodiesterase), and the S cycle (arylsulfatase) were assayed using 0.5 g of air-dried soil (<2 mm). Duplicate replicates and one control were used for all the soils that were tested; furthermore, the appropriate substrate was used for each assay, and reactions were incubated for 1 h at 37°C at their optimal pH as described in [5]. For the controls, the substrate was added after the 1 h incubation period and subtracted from a sample control value. Enzyme activity is expressed in mg p-nitrophenol

Amplicon pyrosequencing (bTEFAP) was originally described by Dowd et al. (2008) and has been utilized in describing a wide range of environmental and health-related microbiomes including the intestinal populations of a variety of sample types and environments, including cattle [16–18]. The 16S universal eubacterial primers (F = AGRGTTTGATCMTGGCTCAG, R = GTNTTACNGCGGCKGC

The sequence data derived from the sequencing analysis was processed using a proprietary analysis pipeline (www.mrdnalab.com, MR DNA, Shallowater, TX). Sequences were depleted of barcodes and primers, and all sequences shorter than <200 bp were removed. Sequences with ambiguous base calls were removed, and

sequences were then denoised and chimeras were removed. Operational taxonomic units were defined after the removal of singleton sequences, clustering at 3% divergence (97% similarity) [16–20]. OTUs were then taxonomically classified using BLASTn against a curated Greengenes database [20] and compiled into each taxonomic level into both "counts" and "percentage" files. Operational taxonomic unit tables (OTU) tables reported by the Molecular Research Facility were used to complete all statistical analysis. Bacterial diversity was estimated by using the

) indexes; both were calculated using the

TGG) were used for PCR amplification. A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds; 53°C for 40 seconds and 72°C for 1 minute; and after which a final elongation step at 72°C for 5 minutes was performed. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt AMPure beads (Agencourt Bioscience Corporation, MA, USA). Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents and follow-

sequences with homopolymer runs exceeding 6 bp were also removed. All

) and Equitability (J<sup>0</sup>

enzymes relevant in C cycling (β-glucosidase), C and N cycling (β-

.

**2.3 Pyrosequencing data processing and analysis**

results as OUT tables.

*Microorganisms*

**2.2 Soil enzyme activities**

(PN) released in kg�<sup>1</sup> soil h�<sup>1</sup>

ing manufacturer's guidelines.

Shannon-Wiener (H<sup>0</sup>

**48**

## **3.1 OTU abundance at the Guánica Dry Forest as affected by sampling period and tree species**

Sequencing data revealed 17 predominant bacterial phyla for this forest (**Table 1** and **Figure 2**). The phyla with the highest relative abundance were *Proteobacteria*


### *Microorganisms*


#### **Table 1.**

*Kruskal-Wallis analysis of the relative abundance (%) of bacteria at the Guánica Dry Forest as affected by wet and dry periods (n = 9).*

(44%) and *Actinobacteria* (37%). These dominant bacterial phyla presented the same pattern during the wet and dry period. During the wet period, relative abundance of *Actinobacteria* decreased almost four times when compared to the dry period, and the relative abundance of *Proteobacteria* almost duplicated (**Table 1** and **Figure 2**). Relative abundance of *Planctomycetes*, *Acidobacteria*, *Gemmatimonadetes*, and *Firmicutes* was 1–2 orders of magnitude higher during the dry period when compared to wet period, whereas only *Proteobacteria* and *Bacteroidetes* were 1–2 orders of magnitude higher during the wet period when compared to the dry period. The most predominant bacterial phyla for all tree species during the wet and

*Nonparametric ANOVA for bacterial alpha diversity in soils as affected by tree species and sampling period at*

*Kruskal-Wallis analysis of the relative abundance (%) of bacteria phyla at the Guánica Dry Forest as affected*

*Bold numbers represent significant differences (p < 0.05).*

*Significant differences are found in bold.*

*by tree species (Ficus citrifolia, Pisonia albida, and Tabebuia heterophylla).*

**Table 2.**

**Table 3.**

**51**

*the Guánica Dry Forest.*

**Bacterial phyla** *F. citri.* **S.D.** *P. albida* **S.D.** *T. heter.* **S.D. p** Tree Species *Bacteroidetes* 3.71 1.27 4.97 3.45 6.92 4.89 0.69 *Chloroflexi* 1.91 1.39 1.29 1.24 1.84 1.19 0.70 *Cyanobacteria* 0.05 0.13 0.06 0.15 0.00 0.00 0.59 *Actinobacteria* 27.95 11.7 29.16 14.5 24.77 13.07 0.85 *Verrucomicrobia* 0.30 0.39 0.25 0.17 0.07 0.14 0.18 *Spam (candidate division)* 0.00 0.00 0.07 0.17 0.04 0.10 0.59 *Planctomycetes* 1.46 1.00 1.65 1.12 1.30 0.96 0.85 *Nitrospirae* 0.53 0.49 0.22 0.17 0.36 0.35 0.65 *Acidobacteria* 1.65 1.39 1.67 1.40 1.20 0.96 0.85 *Gemmatimonadetes* 1.03 0.91 0.59 0.63 1.28 0.80 0.20 *Firmicutes* 3.97 1.75 2.24 1.87 5.51 3.22 0.17 *Tenericutes* 0.04 0.09 0.00 0.00 0.00 0.00 0.37 *ws3 (candidate division)* 0.00 0.00 0.00 0.00 0.16 0.29 0.12 *Proteobacteria* 57.23 17 57.77 15.4 56.51 14.77 0.98 *op3 (candidate division)* 0.06 0.14 0.00 0.00 0.00 0.00 0.37 *tm6 (candidate division)* 0.04 0.09 0.00 0.00 0.01 0.03 0.59 *tm7 (candidate division)* 0.09 0.15 0.07 0.11 0.03 0.07 0.77

*First Insights into the Resilience of the Soil Microbiome of a Tropical Dry Forest…*

*DOI: http://dx.doi.org/10.5772/intechopen.90395*

dry periods were *Proteobacteria*, *Actinobacteria*, and *Bacteroidetes* (**Table 2**). Kruskal-Wallis test did not demonstrate significant differences in bacterial relative

#### **Figure 2.**

*Relative abundance (%) of bacterial phyla at the Guánica Dry Forest during the wet (W) and dry (D) period under three different tree species (F =* Ficus citrifolia*,P=* Pisonia albida*,T =* Tabebuia heterophylla*)*.


*First Insights into the Resilience of the Soil Microbiome of a Tropical Dry Forest… DOI: http://dx.doi.org/10.5772/intechopen.90395*

#### **Table 2.**

*Kruskal-Wallis analysis of the relative abundance (%) of bacteria phyla at the Guánica Dry Forest as affected by tree species (Ficus citrifolia, Pisonia albida, and Tabebuia heterophylla).*

#### **Table 3.**

*Nonparametric ANOVA for bacterial alpha diversity in soils as affected by tree species and sampling period at the Guánica Dry Forest.*

(44%) and *Actinobacteria* (37%). These dominant bacterial phyla presented the same pattern during the wet and dry period. During the wet period, relative abundance of *Actinobacteria* decreased almost four times when compared to the dry period, and the relative abundance of *Proteobacteria* almost duplicated (**Table 1** and **Figure 2**). Relative abundance of *Planctomycetes*, *Acidobacteria*, *Gemmatimonadetes*, and *Firmicutes* was 1–2 orders of magnitude higher during the dry period when compared to wet period, whereas only *Proteobacteria* and *Bacteroidetes* were 1–2 orders of magnitude higher during the wet period when compared to the dry period. The most predominant bacterial phyla for all tree species during the wet and dry periods were *Proteobacteria*, *Actinobacteria*, and *Bacteroidetes* (**Table 2**). Kruskal-Wallis test did not demonstrate significant differences in bacterial relative

**Figure 2.**

**50**

*Relative abundance (%) of bacterial phyla at the Guánica Dry Forest during the wet (W) and dry (D) period under three different tree species (F =* Ficus citrifolia*,P=* Pisonia albida*,T =* Tabebuia heterophylla*)*.

**Phyla Dry S.D. Wet S.D. p value**

*Acidobacteria* **2.28 1.18 0.73 0.60 0.01** *Gemmatimonadetes* **1.48 0.75 0.45 0.44 0.01** *Firmicutes* **5.32 2.86 2.49 1.37 0.01** *Tenericutes* 0.00 0.00 0.02 0.07 >0.9999 *ws3 (candidate division)* 0.11 0.24 0.00 0.00 0.47 *Proteobacteria* **44.35 5.87 69.99 7.78 <0.0001** *op3 (candidate division)* 0.04 0.12 0.00 0.00 >0.9999 *tm6 (candidate division)* 0.02 0.07 0.01 0.02 >0.9999 *tm7 (candidate division)* 0.05 0.10 0.08 0.13 0.86

*Kruskal-Wallis analysis of the relative abundance (%) of bacteria at the Guánica Dry Forest as affected by wet*

*Bold numbers represent significant differences (p < 0.05).*

**Table 1.**

*Microorganisms*

*and dry periods (n = 9).*

**Bacterial phyla**


**3.3 Relationship between the relative abundance of taxa and enzyme activities**

*Canonical correspondence analysis (CCA) of bacterial phyla demonstrating the effect of wet/dry periods at the Guánica Dry Forest. Blue symbols represent wet periods and red symbols represent dry periods. Triangles, circles and plus sign represent tree species (Tabebuia heterophylla, Ficus citrifolia and Pisonia albida), respectively.*

*Canonical correspondence analysis (CCA) of bacterial phyla and soil enzyme activities*

*(Phosdi = phosphodiesterasae, ArylSul = aryl sulphatase, APho = alkaline phosphatase, AcidPho = acid phosphatase, Bcosi = β-glucosaminidase, and Bglu = β-glucosidase). Vectors represent enzyme activities.*

Axis 1 of the canonical correspondence analysis for bacterial community explained 80% of the variation (**Figure 3**). Two groups were segregated with regard

*First Insights into the Resilience of the Soil Microbiome of a Tropical Dry Forest…*

*DOI: http://dx.doi.org/10.5772/intechopen.90395*

**Figure 3.**

**Figure 4.**

**53**

#### **Table 4.**

*Bacterial indicator species analysis (ISA) at the Guánica Dry Forest.*

abundance due to tree species (**Table 2**). There were no significant differences in any of the diversity indexes with regard to tree species (**Table 3**). Sampling period exhibited an effect on bacterial species richness (P = 0.05) and on equitability (P = 0.0243). but not on the Shannon index. We found higher bacterial species richness during the wet period and higher equitability during the dry period (**Table 3**).

#### **3.2 Evaluation of indicator species analysis (ISA) for this forest ecosystem**

The identification of species associated or indicative of groups of samples is a common aspect of ecological research [24]. Indicator species analysis (ISA) identified several bacterial (**Table 4**) species responsible for changes in soil microbial communities. Out of 185 bacterial OTUs, 10 served as indicator species for *Ficus citrifolia* during the wet period (**Table 4**). A total of 31 bacterial OTUs were identified as indicator species for *Pisonia albida* during the wet period. No bacterial indicator species were found for the dry period (**Table 4**).

*First Insights into the Resilience of the Soil Microbiome of a Tropical Dry Forest… DOI: http://dx.doi.org/10.5772/intechopen.90395*
