World Journal of Microbiology and Biotechnology (2018) 34:58 https://doi.org/10.1007/s11274-018-2436-5 ORIGINAL ARTICLE Moss habitats distinctly affect their associated bacterial community structures as revealed by the high-throughput sequencing method Su Wang1 · Jing Yan Tang1 · Jing Ma1 · Xue Dong Li1 · Yan Hong Li1 Received: 23 September 2017 / Accepted: 17 March 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract To better understand the factors that influence the distribution of bacteria associated with mosses, the communities inhabit- ing in five moss species from two different habitats in Beijing Songshan National Nature Reserve were investigated using the high-throughput sequencing method. The sequencing was performed based on the bacterial 16S rRNA and 16S rDNA libraries. Results showed that there are abundant bacteria inhabiting in all the mosses sampled. The taxonomic analysis of these bacteria showed that they mainly consisted of those in the phyla Proteobacteria and Actinobacteria, and seldom were from phylum Armatimonadetes, Bacteroidetes and Firmicutes. The hierarchical cluster tree, based on the OTU level, divided the bacteria associated with all samples into two branches according to the habitat types of the host (terrestrial and aquatic). The PCoA diagram further divided the bacterial compositions into four groups according to both types of habitats and the data sources (DNA and RNA). There were larger differences in the bacterial community composition in the mosses col- lected from aquatic habitat than those of terrestrial one, whether at the DNA or RNA level. Thus, this survey supposed that the habitat where the host was growing was a relevant factor influencing bacterial community composition. In addition, the bacterial community detected at the RNA level was more sensitive to the habitat of the growing host, which could also be proved by the significantly differences in the predicted function by PICRUSt and the metabolically active dominant genera between different groups. This study expands the knowledge about the interactions between mosses and microbes. Keywords Bacterial composition · Aquatic and land mosses · High-throughput DNA sequencing Introduction circulation, and are also sensitive to environmental pollu- tion by heavy metals (Zhou et al. 2017). In the evolution of the plant kingdom, bryophytes represent Mosses, a type of bryophyte, are non-vascular, small plants that transitioned from aquatic to terrestrial environ- green plants with very simple structures. Endophytic bacte- ments, thus they belong to a relatively small group in the ria are known to colonize mosses, and studies have shown higher plants. There are more than 20,000 species of bryo- that they may play an important role in enhancing the envi- phytes in the world (http://www.thepl antli st.org; Shaw and ronment adaptability of mosses to cope with different kinds Renzaglia 2004). Bryophytes are widely distributed and well of abiotic and biotic stresses (e.g., temperature fluctuations, adapted to extreme environmental conditions; they function UV-B radiation exposure, desiccation stress, defense against primarily to conserve soil and water and enhance water pre-existing microorganisms) (Opelt and Berg 2004; Opelt et al. 2007a, b, c; Liu et al. 2014). Epiphytic bacteria have also been observed attached tightly to the surface of some Electronic supplementary material The online version of this mosses (Ma et al. 2017) and are believed to provide protec- article (https ://doi.org/10.1007/s1127 4-018-2436-5) contains tion from freezing for the Antarctic moss Bryum argenteum supplementary material, which is available to authorized users. (Raymond 2016). Therefore, both the endophytic and epi- phytic bacteria associated with mosses may play important * Yan Hong Li [email protected] roles in their adaptation mechanisms. Although several studies have been carried out to exam- 1 College of Life Science, Capital Normal University, ine the bacterial diversity and community composition asso- Xisanhuan North Road 105#, Haidian District, ciated with different mosses (Liu et al. 2014; Koua et al. Beijing 100048, China Vol.:(0123456789)1 3 58 Page 2 of 13 World Journal of Microbiology and Biotechnology (2018) 34:58 2015; Tang et al. 2016; Tian and Li 2016; Ma et al. 2017), locations (representing two different habitat types: terres- many factors related to this association remain unclear. In trial and aquatic) to investigate their associated bacterial a survey of bacteria associated with ten liverworts and ten community, using the high-throughput method, based on mosses in Tibet (Tang et al. 2016), it was supposed that 16S rDNA and 16S rRNA libraries. Our objective was to host phylogeny would have a strong influence on the asso- offer further insight on the influence of habitat and host ciated bacterial community, and that niche also played an phylogeny on the bacterial community composition in important role when the hosts were phylogenetically more mosses, and to provide useful information on the interac- similar. Recent research (Ma et al. 2017) of bacterial com- tions between mosses and microbes. munities in four moss species showed that the associated bacteria were mainly correlated with differences in moss species resources, although the bacterial community compo- sition also varied with the sampling season and data source. Materials and methods We suppose that the reason for the differing results is that the experiments were focused on a variety of purposes. In Sample collection and processing the first survey, all samples were collected from nearly the same niches, whereas the second survey involved two dif- Mosses were collected from eight different locations in ferent local niches. Thus, the role of niche and phylogenetic Beijing Songshan National Nature Reserve, China, on Oct status in determining the bacterial community composition, 9th, 2015. They were named as sample no. 1, 2, 3, 5, 6, and how bacteria interact or respond to changes in the hosts, 9, 10, and 11, according to the locations (see Table 1), remains unknown. The interactions between a plant and its respectively. Due to the small volume and low weight of microbe system are very complicated, primarily because the each moss species, about 3 g of each sample containing bacteria associated with the hosts are comprised of a mixed hundreds of moss individuals were sampled and mixed population, many of which are uncultivated. In addition, the into one composite sample used for the next study. They hosts are complex organisms and their growth is influenced were stored at 4 °C and transferred into the laboratory in by many environmental factors. Thus, it is difficult to know 2 h once they were collected. They were first treated by how bacteria interact with their hosts and whether their com- removing the attached matrix, followed by several times position is influenced by various factors. rinsing with sterile water to clean the mosses. After that, In order to better understand the factor which influ- the plants were soaked in 70% ethanol for 3 min, then ences on the associated bacterial community composition washed with sterile water five times, and finally dried with in the mosses, five moss species (with differing phylo- filter paper. genetic relationships) were collected from eight different Table 1 Sampling information for all samples collected Sample ID Plant species Taxonomy Sample location Altitude (m) Tem- Humidity (%) Sample habitat charac- perature teristics (oC) 1 Cratoneuron filicinum Hypnales N: 40°30′26.08″ 758 3 85 Rocks in a stream (Hedw.) Spruce Amblystegiaceae E: 115°48′55.38 2 Haplocladium micro- Hypnales N: 40°30′25.36″ 758 3 85 Rocks in a stream phyllum (Hedw.) Broth Thuidiaceae E: 115°48′55.79″ 3 Pylaisiella polyantha Hypnales N: 40°30′31.35″ 769.1 9 30 Rocks far from the stream (Hedw.) Grout Hypnaceae E: 115°48′56.26″ 5 Pylaisiella polyantha Hypnales N: 40°30′36.31″ 798.6 9 30 Rocks far from the stream (Hedw.) Grout Hypnaceae E: 115°48′59.01″ 6 Campyliadelphus Hypnales N: 40°30′39.96″ 819.1 3 87 Rocks in a stream polygamum (B. S. G.) Amblystegiaceae E: 115°49′2.28″ Kanda 9 Bryum algovicum Sendt. Bryales N: 40°30′52.65″ 840 9 25 Rocks far from the stream Bryaceae E:115°49′07.59″ 10 Pylaisiella polyantha Hypnales N: 40°30′52.83″ 843.2 9 25 Rocks far from the stream (Hedw.) Grout Hypnaceae E: 115°49′07.81″ 11 Cratoneuron filicinum Hypnales N: 40°30′51.63″ 843.2 4 80 Bank of the stream (Hedw.) Spruce Amblystegiaceae E: 115°49′07.45″ 1 3 World Journal of Microbiology and Biotechnology (2018) 34:58 Page 3 of 13 58 Total DNA extraction and amplification dNTPs, 0.8 µL Forward Primer 926F (5 µM), 0.8 µL of the bacterial 16S rDNA Reverse Primer 1392R (5 µM), 0.4 µL FastPfu Polymer- ase] and 10 ng of template DNA. To distinguish the differ- Total DNA of each moss sample was extracted to be used ent samples and detection levels, a barcoded-tag with eight for the bacterial analysis based on 16S rDNA sequences. nucleotide bases were randomly added to the upstream of Approximately 1 g of each surface treated sample (including the universal primers. The thermal cycling conditions were about 50 individuals) was ground in liquid nitrogen, and the as follows: an initial denaturation at 95 °C for 3 min, and total DNA was extracted using the FastDNA SPIN Kit for 27 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for Soil (MPBio, USA), according to the instructions provided 45 s, with a final extension at 72 °C for 10 min. Amplicons by the manufacturer. Then PCR amplification was performed derived from three repeats each sample were mixed together, using a Taq Mix (2 × Taq PCR Master Mix, Biomed) in a extracted from 2% agarose gels and purified using the Axy- total volume of 50 µL, which contained 25 µL Taq Mix, Prep DNA Gel Extraction Kit (Axygen Biosciences, USA) 20 µL ddH2O, 2 µL of primer 799F (5′-AACMGGA TTA according to the manufacturer’s instructions and quantified GAT ACC CTG-3′) and 1492R (5′-GGT TAC CTT GTT ACG using QuantiFluor™-ST (Promega, USA).