Soil bacterial community structure in five tropical forests in Malaysia and one temperate forest in Japan revealed by pyrosequencing analyses of 16S rRNA gene sequence variation

Abstract

Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40-50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40-60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro-and macro-environments, but macroenvironmental diversity made a larger contribution to total bacterial diversity in forest soil.

Geographical variation in soil bacterial community structure in tropical forests in Southeast Asia and temperate forests in Japan based on pyrosequencing analysis of 16S rRNA

“…This extends our previous study (Miyashita et al, 2013) by including an additional 21 tropical forests in Malaysia, Indonesia and Singapore, and one temperate forest in Japan. These 28 forests had different aboveground vegetation, including dipterocarp forest, acacia plantation, oil palm plantation, limestone forest and mangrove.…”

“…Occurrence of OTUs within and between investigated forests Based on the concerted evolution and conserved copy number variation of bacterial 16S rRNA cistrons, the number of 16S rRNA sequence reads per OTU may be regarded as the representative of the number of individual bacteria per species (OTU) in the forest soils investigated, although it is difficult to convert the number of sequence reads to the exact number of individual bacteria (Miyashita et al, 2013;Miyashita, 2015). For the total data, the most abundant OTU, which belonged to Acidobacteria subgroup Gp1, represented approximately 3% (7,934) of all reads (256,693), and the second most abundant OTU, which belonged to Acidobacteria subgroup Gp2, represented approximately 2% of reads (5,384) ( Table 3).…”

“…PCR amplification of bacterial 16S rRNA region Extracted microbial DNA was used as template for PCR amplification of part of the 16S rRNA region as described (Miyashita et al, 2013;Miyashita, 2015). Briefly, two PCR primers were used for reactions in this study: 9F 5'-GAGTTTGATCCTGGCTCAG-3' and 533R 5'-TIACC-GIIICTICTGGCAC-3'.…”

“…These primers amplify a 510-bp fragment that includes three variable regions (V1, V2 and V3) of the 16S rRNA gene in bacteria, archaea and eukarya (Baker et al, 2003). To distinguish the 28 samples from each other, a unique tag sequence was attached to the 5' end of the forward and reverse primers, as described (Miyashita et al, 2013). The PCR solution (50 μl) contained 0.2 mM dNTPs, 2.5 units of TaKaRa ExTaq (Takara Bio, Otsu, Japan), 10 × Taq buffer, 2 mM MgCl 2 , 0.2 μM each of the forward and reverse primers, and ~10 ng of template DNA.…”

“…Recently, by studying 25 temperate grassland sites from four continents (Africa, Australia, Europe and North America) by high-throughput sequencing of 16S rRNA (bacteria and archaea) and an ITS region (fungi), it was shown that plant α-diversity was poorly related to that for microbial groups (Prober et al, 2015). In our previous work on five tropical forests in Malaysia and one temperate forest in Japan, using pyrosequencing analy-ses of bacterial 16S rRNA (Miyashita et al, 2013), we also observed a similar composition of higher taxonomic taxa and similar bacterial species diversity in the six forests.…”