**2. Methods**

### **2.1 Study site**

The study was located in the coastal association of the Guánica Dry Forest latitude 66°53′30″W longitude 17°58′0″N [16–18]. In this area of the forest, dwarf individual adult trees from different species are separated by exposed rocks which prevent overlap among trees forming "monospecific tree islands" (**Figure 1A** and **B**). These "monospecific tree islands" have little or no mineral soil (**Figure 1A**), with a highly organic substrate composed of shallow monospecific litter and humus (**Figure 1C** and **D**), which varies according to variations in the ground relief and season from 2 to 8 cm depth [16]. These characteristics make this forest an ideal system to study tree species effects as there are no confounding effects of overlapping roots from other species. We chose three trees from three species, previously tagged and studied, that grow from 100 m to approximately 300 m from the coast.

**101**

**Figure 1.**

*and dry adventitious roots.*

*Tree Species and Precipitation Effect on the Soil Microbial Community Structure and Enzyme…*

The three tree species selected are: *Tabebuia heterophylla* (*DC.*) Briton, Ann, a facultative deciduous species*; Pisonia albida* (Heimerl) Briton ex Scandal, an obligate deciduous species and *Ficus citrifolia* Mill., Gard., a facultative deciduous species. The trees are distributed more or less randomly across this area and are dominant species of the coastal region [17], no spatial distribution has been detected. During this study, the highest precipitation was reported during the month of August 2011 (212 mm) and the lowest precipitation was reported for the month of October 2011 (85.09 mm). The total accumulated rainfall was 738 mm.

*Detailed description of study site. (A) Tree species that has established its growth in the cracks and crevices of the calcareous rock. Black arrow demonstrates a new soil pocket that is being formed as a by-product of the decomposition of the tree's litter. (B and C)* Tabebuia heterophylla *tree that has established on the calcareous rock at the Guánica Dry Forest; here, we can observe small litter pockets that have formed in the cracks of the rock. (D) Figure demonstrates that the substrate collected is humus with a vast quantity of fragmented litter,* 

Total organic matter, soil texture, pH and moisture analyses were completed. Soil total organic matter was determined by the ignition method. Soil texture was determined by the pipette method [19]. Soil pH was determined in a 1:5 (soil:water) mixture using an Orion 420A pH Meter [20]. Soil moisture was determined by

Microbial community structure was evaluated by the ester-linked (EL)-FAME method as described by [21]. A total of 3 g of field moist soil was used for each sample, and four steps were completed: (a) saponification and methylation of ester-linked fatty acids were performed by incubating the 3 g of soil in 15 ml of 0.2 M KOH in methanol at 37°C during 1 h—samples were vortexed every 10 min during the

*DOI: http://dx.doi.org/10.5772/intechopen.82579*

**2.2 Selected soil substrate analyses**

**2.3 EL-FAME analysis**

weighing and drying 1 g of soil at 105°C during 24 h [20].

*Tree Species and Precipitation Effect on the Soil Microbial Community Structure and Enzyme… DOI: http://dx.doi.org/10.5772/intechopen.82579*

#### **Figure 1.**

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

enzyme activity [10].

environments such as tropical dry forest.

activity [9] and rainfall pulses may result in pulses of microbial growth which may lead to pulsed secretion of soil enzymes promoting a temporary increase of soil

Understanding the dynamics of forest soil ecosystem depends on elucidating the contribution of individual plant species to the soil biota and the process that they regulate [11]. Determining and quantifying plant species effects under natural conditions can be difficult due to environmental noise and the interaction among species present in an area [12]. Little is known about the effects of dominant vegetation on the soil biogeochemical processes such as enzyme activities [13]. Even less is known about the plant-soil-microbial interactions that take place in extreme

Nearly 42% of tropical forests around the world are seasonally dry plant communities, where around half of the Central American and Caribbean land area is characterized by a tropical or subtropical dry forest climate. In the Coastal Plateau of the Guánica Dry Forest (GDF), an UNESCO/MAB Biosphere Reserve, dwarfed trees grow isolated from one another in the cracks of the calcareous platform, forming individual islands of fertility [14]. A lack of interspecific competition is observed, as tolerance to environmental stress and scarcity of space for establishment make it difficult for aboveground and belowground competition, also contributing to the evenness of tree species found in this area [14]. The substrate is derived from limestone made from marine deposits that vary throughout the forest from deep alluvial fans to exposed fractured limestone with shallow soil pockets [15]. This naturally occurring plant community provides the ideal conditions to determine how specific tree species affect the soil microbial community composition and enzymatic potential in a dry forest. In order to understand how trees impact soil microbial communities, a five-month study was conducted at the GDF. We selected three dwarfed, isolated tree species (a pantropical species and two native species) that are highly distributed among the forest, hypothesizing that these trees may harbour different microbial community structure and activities. The tree species selected complied with the following requirements: (1) that trees were growing in cracks isolated from other trees by exposed rock and (2) that their litter and belowground substrate originated from their own residue decomposition [16]. Additionally, this forest experiences bimodal and pulsed precipitation patterns [17, 18] that may contribute in the alteration of the microbial dynamics and nutrient turnover of the forest. Our objectives were: (1) to determine if tree species traits had an effect on the soil microbial community structure and activities and (2) to determine the effects of sampling period on the soil microbial community structure and activities.

The study was located in the coastal association of the Guánica Dry Forest latitude 66°53′30″W longitude 17°58′0″N [16–18]. In this area of the forest, dwarf individual adult trees from different species are separated by exposed rocks which prevent overlap among trees forming "monospecific tree islands" (**Figure 1A** and **B**). These "monospecific tree islands" have little or no mineral soil (**Figure 1A**), with a highly organic substrate composed of shallow monospecific litter and humus (**Figure 1C** and **D**), which varies according to variations in the ground relief and season from 2 to 8 cm depth [16]. These characteristics make this forest an ideal system to study tree species effects as there are no confounding effects of overlapping roots from other species. We chose three trees from three species, previously tagged and studied, that grow from 100 m to approximately 300 m from the coast.

**100**

**2. Methods**

**2.1 Study site**

*Detailed description of study site. (A) Tree species that has established its growth in the cracks and crevices of the calcareous rock. Black arrow demonstrates a new soil pocket that is being formed as a by-product of the decomposition of the tree's litter. (B and C)* Tabebuia heterophylla *tree that has established on the calcareous rock at the Guánica Dry Forest; here, we can observe small litter pockets that have formed in the cracks of the rock. (D) Figure demonstrates that the substrate collected is humus with a vast quantity of fragmented litter, and dry adventitious roots.*

The three tree species selected are: *Tabebuia heterophylla* (*DC.*) Briton, Ann, a facultative deciduous species*; Pisonia albida* (Heimerl) Briton ex Scandal, an obligate deciduous species and *Ficus citrifolia* Mill., Gard., a facultative deciduous species. The trees are distributed more or less randomly across this area and are dominant species of the coastal region [17], no spatial distribution has been detected. During this study, the highest precipitation was reported during the month of August 2011 (212 mm) and the lowest precipitation was reported for the month of October 2011 (85.09 mm). The total accumulated rainfall was 738 mm.

#### **2.2 Selected soil substrate analyses**

Total organic matter, soil texture, pH and moisture analyses were completed. Soil total organic matter was determined by the ignition method. Soil texture was determined by the pipette method [19]. Soil pH was determined in a 1:5 (soil:water) mixture using an Orion 420A pH Meter [20]. Soil moisture was determined by weighing and drying 1 g of soil at 105°C during 24 h [20].

#### **2.3 EL-FAME analysis**

Microbial community structure was evaluated by the ester-linked (EL)-FAME method as described by [21]. A total of 3 g of field moist soil was used for each sample, and four steps were completed: (a) saponification and methylation of ester-linked fatty acids were performed by incubating the 3 g of soil in 15 ml of 0.2 M KOH in methanol at 37°C during 1 h—samples were vortexed every 10 min during the

incubation period and, after the incubation period was completed, 3 ml of 1.0 acetic acid was added to neutralize the mixture's pH; (b) partitioning of the FAMEs into an organic phase was achieved by adding 10 ml of hexane and centrifuging the preparations at 480×*g* for 10 min; (c) the hexane layer was transferred to a clean glass tube and evaporated under a N2 stream and (d) FAMEs were suspended in 0.5 ml of 1:1 hexane:methyl-tert butyl ether and transferred to GC vials for analysis. Extractions were analysed as described by [4]. A 6890 GC Series II (Hewlett Packard, Wilmington, DE, USA) equipped with a flame ionization detector and fused silica capillary column (25 m × 0.2 mm) with ultra-high purity H2 as the gas carrier was used to analyse the extractions. The temperature program was ramped from 170 to 250°C at 5°C min<sup>−</sup><sup>1</sup> as described. The fatty acids were identified and quantified by comparing the retention times and peak areas to MIDI standards. The MIDI software provides FAME relative peak areas (percentage) based on the total FAMEs in a sample (based on the Aerobe method of the MIDI system). FAME concentrations (nmol g<sup>−</sup><sup>1</sup> soil) were calculated by comparing peak areas to an analytical standard (19:0, Sigma Chemical Co., St. Louis, MO) calibration curve. The FAMEs are described by the number of C atoms, followed by a colon, the number of double bonds and then by the position of the first double bond from the methyl (ω) end of the molecule. Cis isomers are indicated by c and branched fatty acids are indicated by the prefixes *i* and *a* for iso and anteiso, respectively. Other notations are Me for methyl, OH for hydroxy and cy for cyclopropane.
