Pyrosequencing analysis of a bacterial community associated with lava-formed soil from the Gotjawal forest in Jeju, Korea

Abstract

In this study, we analyzed the bacterial diversity in soils collected from Gyorae Gotjawal forest, where globally unique topography, geology, and ecological features support a forest grown on basalt flows from 110,000 to 120,000 years ago and 40,000 to 50,000 years ago. The soils at the site are fertile, with rocky areas, and are home to endangered species of plants and animals. Rainwater penetrates to the groundwater aquifer, which is composed of 34% organic matter containing rare types of soil and no soil profile. We determined the bacterial community composition using 116,475 reads from a 454-pyrosequencing analysis. This dataset included 12,621 operational taxonomic units at 3% dissimilarity, distributed among the following groups: Proteobacteria (56.2%) with 45.7% of α-Proteobacteria, Actinobacteria (25%), Acidobacteria (10.9%), Chloroflexi (2.4%), and Bacteroidetes (0.9%). In addition, 16S rRNA gene sequences were amplified using polymerase chain reaction and domain-specific primers to construct a clone library based on 142 bacterial clones. These clones were affiliated with the following groups: Proteobacteria (56%) with 51% of α-Proteobacteria, Acidobacteria (7.8%), Actinobacteria (17.6%), Chloroflexi (2.1%), Bacilli (1.4%), Cyanobacteria (2.8%), and Planctomycetes (1.4%). Within the phylum Proteobacteria, 56 of 80 clones were tentatively identified as 12 unclassified genera. Several new genera and a new family were discovered within the Actinobacteria clones. Results from 454-pyrosequencing revealed that 57% and 34% of the sequences belonged to undescribed genera and families, respectively. The characteristics of Gotjawal soil, which are determined by lava morphology, vegetation, and groundwater penetration, might be reflected in the bacterial community composition.

Keywords: 16S rRNA gene; Gotjawal forest soil; bacterial diversity; pyrosequencing.

Figure 1

(A) A map of Jeju Island showing the Gotjawal distribution (blue line) and the sampling site (blue triangle symbol). (B) Photographs showing the Gyorae Gotjawal forest in Jeju (N 33° 26′ 24.9″ and E 126° 39′ 44.5″, Korea). Pahoehoe lava and a'a lava were mixed when the lava flowed. The lava-formed forest was developed, with characteristic topography including lava domes and depressions. Vegetation in this area is mainly deciduous broadleaf trees, Styrax japonicus, Zelkova serrata, Thunb Makino, and Acer palmatum Thunb. ex Murray (Maple). Within the herb layer, Arachniodes standishii (T. Moore) Ohwi is the predominant species.

Figure 2

Rarefaction curves of 454-pyrosequences indicating the observed number of OTUs in the Gyorae Gotjawal forest soil.

Figure 3

Phylogenetic distribution of operational taxonomic units (OTUs) and 454-pyrosequencing/clones observed in Gotjawal soil.

Figure 4

Phylogenetic tree of 142 bacterial clone sequences. (A) Proteobacteria from Gotjawal soils (80 clone sequences), with reference sequences. Twelve previously unknown clades are indicated as novel genera within the phylum. (B) Acidobacteria (13 clone sequences), with reference sequences. (C) Actinobacteria (28 clone sequences). (D) Chloroflexi, Bacilli, Cyanobacteria, and Planctomycetes (10 clone sequences). Bold letters refer to clone sequences. Each clade that appears to be a previously discovered taxon, based on the 16S rRNA divergence from the nearest previously known relative, is indicated.