Changes of soil microbial community composition on rock surface after the colonization of lithophytic bryophytes
The average number of microorganisms in the rock surface of the bryophytes was 22340 (Hyophila involute 22063, Eurohypnum leptothallum 22779, Didymodon constrictus 22180 ), 22329 in the exposed rock surface and 19925 in the original control soil.These microorganisms mainly include Fungi, Bacteria, Eukaryota, Archaea, Viruses. At the phylum level, bacteria had the largest number of species, followed by Eukaryota, Archaea, Fungi and Viruses ( Fig.1-A ).The top 10 phylum in the abundance of Bacteria were Actinobacteria、Proteobacteria、Acidobacteria、Cyanobacteria、Chloroflexi、Candidatus_Rokubacteria、Gemmatimonadetes_d_Bacteria、unclassified_d__Bacteria、Bacteroidetes、Planctomycetes(Fig.1-B).The top 10 phylum in the abundance of Archae were Euryarchaeota、Thaumarchaeota、unclassified_d__Archaea、Candidatus_Bathyarchaeota、Crenarchaeota、Candidatus_Woesearchaeota、Candidatus_Thorarchaeota、Candidatus_Micrarchaeota、Candidatus_Korarchaeota、Candidatus_Altiarchaeota(Fig.1-C).The top 10 phylum in the abundance of Eukaryot were Streptophyta、Chordata、Ascomycota、Arthropoda、Mucoromycota、Bacillariophyta、unclassified_d_Eukaryota、Nematoda、Basidiomycota、Chytridiomycota(Fig.1-D)。The top 10 phylum in the abundance of Viruses were Artverviricota、unclassified_d__Viruses、Nucleocytoviricota、Phixviricota、Uroviricota(Fig.1-E)。The top 10 phylum in the abundance of Fungi were Mucoromycota、Basidiomycota、Chytridiomycota、Zoopagomycota、Microsporidia、Cryptomycota、Blastocladiomycota、Ascomycota(Fig.1-F)。
At the species level, Fungi 155, Bacteria 18460, Eukaryota 459, Archaea 395, Viruses 63 in the original control soil, and Fungi 181, Bacteria 20597, Eukaryota 631, Archaea 455, Viruses 94 in the exposed rock surface(Fig.2-A). The average species of various microorganisms in the rock surface habitat after the colonization of lithophytic bryophytes were Fungi 235, Bacteria 20535, Eukaryota 816, Archaea 503, Viruses155, which were more than those in the bare rock surface habitat. Compared with the original control soil, the growth of various microorganisms was Fungi 52 %, Bacteria 11 %, Eukaryota 78 %, Archaea 27 %, Viruses 146 %. Among them, Viruses had the largest growth of 146 %, followed by Eukaryota, Fungi, Archaea, and Bacteria had the smallest growth. We can see that the number of various microorganisms in the rock surface increased significantly after the colonization of bryophytes, and the impact on Viruses, Eukaryota and Fungi was greater than that of Bacteria and Archaea.
The top 15 species of Bacteria in each treatment group were Actinobacteria_bacterium、Acidobacteria_bacterium、Chloroflexi_bacterium、Acidimicrobiia_bacterium、Deltaproteobacteria_bacterium、Candidatus_Rokubacteria_bacterium、Smaragdicoccus_niigatensis、Alphaproteobacteria_bacterium、Solirubrobacterales_bacterium、Archangium_gephyra、Acidimicrobiaceae_bacterium、Gemmatimonadetes_bacterium、Betaproteobacteria_bacterium、Nocardiaceae_bacterium_YC2-7、Microcoleus_sp._PCC_7113(Fig.2-B). The top 15 species of Archaea in each treatment group were Euryarchaeota_archaeon、Thaumarchaeota_archaeon、Candidatus_Bathyarchaeota_archaeon、uncultured_archaeon、Nitrososphaeraceae_archaeon、Nitrosopumilales_archaeon、archaeon_HR01、ANME-2_cluster_archaeon、Thermoplasmata_archaeon、Candidatus_Methanoperedens_nitroreducens、Methanosarcinales_archaeon、Candidatus_Woesearchaeota_archaeon、Hadesarchaea_archaeon、Candidatus_Poseidoniales_archaeon、archaeon(Fig.2-C). The top 15 species of Eukaryota in each treatment group were Physcomitrium_patens、Marchantia_polymorpha、Pseudocrossidium_replicatum、Solanum_lycopersicum、Homo_sapiens、Lupinus_albus、Eucalyptus_grandis、Syntrichia_ruralis、Candida_albicans、Mus_musculus、Pohlia_nutans、Fusarium_oxysporum、Syntrichia_filaris、Beta_vulgaris、Phaeodactylum_tricornutum(Fig.2-D). The top 15 species of Fungi in each treatment group were Candida_albicans、Fusarium_oxysporum、Powellomyces_hirtus、Fusarium_odoratissimum、Rhinocladiella_mackenziei、Parasitella_parasitica、Oidiodendron_maius、Diversispora_epigaea、Lipomyces_starkeyi、Smittium_culicis、Absidia_glauca、Rhizopus_delemar、Gigaspora_rosea、Spizellomyces_sp._'palustris'、Rhizophagus_clarus(Fig.2-E)。The top 15 species of Viruses in each treatment group were Murine_leukemia_virus、Rhodococcus_phage_REQ1、Circular_genetic_element_sp.、Gordonia_phage_Phendrix、Gordonia_phage_Mollymur、Caudovirales_GX15bay、Streptomyces_phage_Henoccus、Gordonia_virus_Suzy、Mycobacterium_phage_Kumao、Arthrobacter_phage_Shoya、Microbacterium_phage_Rasovi、Streptomyces_phage_Yara、Microbacterium_phage_FuzzBuster、Gordonia_virus_Ghobes、Arthrobacter_virus_Joann(Fig.2-F)。
4.2 The main functions of soil microorganisms in the lithophytic bryophytes rock surface
By conducting functional clustering analysis on the overall gene set and selecting the top 50 ranked functions, we found that the main functions of these microorganisms are amino acid metabolism, organic acid metabolism, Metabolism of carbon, nitrogen, phosphorus, sulfur, and methane, Photosynthesis related functions, Secondary metabolite related function, glycometabolism, Enzyme related functions(Pantothenate and CoA biosynthesis) (Fig.3-A).
There are differences in microbial functions between different treatment groups. The difference in microbial functions within Hyophila involute and Eurohypnum leptothallum is small, but they differ greatly from Didymodon constrictus and the control group. However, the difference between Didymodon constrictus and the control group is small (Fig.3-B). It indicated that bryophytes had a significant role in promoting the functional development of rock surface soil, but there were interspecific differences in this contribution.
Effects of lithophytic bryophytes on carbon fixation, nitrogen fixation and phosphorus metabolism functional microorganisms in rock surface
After planting on the rock surface, the microorganisms related to carbon fixation in the soil were 2779, nitrogen fixation 1502, and phosphorus metabolism 1750.In the original control soil, carbon-fixing microorganisms 2031, nitrogen-fixing 1008, and phosphorus metabolism 1141. Carbon-fixing microorganisms 2444, nitrogen-fixing 1288, and phosphorus metabolism 1490 in the bare rock surface habitat. Compared with the original control soil, the growth rate of carbon-fixing microorganisms, nitrogen fixation and phosphorus metabolism in the soil after bryophyte planting rock surface increased by 37 %, 49 % and 53 %, respectively. Compared with the original soil, the growth rate of carbon-fixing microorganisms in the exposed rock surface was 20 %, nitrogen fixation was 28 %, and phosphorus metabolism was 31 % ( Fig.4-A ). We can see that the number of microorganisms related to carbon fixation, nitrogen fixation and phosphorus metabolism in the habitat was significantly increased after the colonization of the rock surface.
Among the top 15 species of microbial abundance related to carbon fixation, nitrogen fixation and phosphorus metabolism, 9 kinds of microorganisms appeared in the three functional groups and accounted for a relatively high proportion, which were respectively:Actinobacteria_bacterium、Acidobacteria_bacterium、Acidimicrobiia_bacterium、Chloroflexi_bacterium、Smaragdicoccus_niigatensis、Solirubrobacterales_bacterium、Deltaproteobacteria_bacterium、Alphaproteobacteria_bacterium、Gemmatimonadetes_bacterium. The unique microbial species in the carbon fixation functional group are Iamiaceae_bacterium_SCSIO_58843、Candidatus_Rokubacteria_bacterium、Betaproteobacteria_bacterium、Thermoleophilia_bacterium(Fig.4-B). The unique microorganisms in the functional groups related to phosphorus metabolism are Thermoleophilia_bacterium、Gaiella_occulta、Archangium_gephyra(Fig.4-C). The unique microorganisms in the nitrogen-fixing functional group are Candidatus_Rokubacteria_bacterium、Gaiella_occulta、Nocardiaceae_bacterium_YC2-7、Betaproteobacteria_bacterium、Microcoleus_sp._PCC_7113、Rhizobiales_bacterium(Fig.4-D).
Functional analysis of microbial carbon fixation, nitrogen fixation and phosphorus metabolism in the habitat of lithophytic bryophytes after planting rock surface
There are differences in the metabolic pathways related to carbon fixation between the moss colonized rock surface and the exposed rock surface compared to the original soil (Fig.5).There are also interspecific differences among lithophytic bryophytes. The carbon fixation metabolic pathways between Hyophila involute and Eurohypnum leptothallum ,Didymodon constrictus are significantly different, while the carbon fixation metabolic pathways between Didymodon constrictus and exposed rock surface habitats are similar. The top 15 metabolic pathways related to carbon fixation were significantly different among the treatment groups. The abundance of metabolic pathways K01681, K01903, K01961, K00031, K01007, K01595, and K01847 in the habitat after the colonization of bryophytes on the rock surface was significantly higher than that in the original control soil. However, the abundance of K01848, K00174, K00297, K00175 and K02446 decreased significantly ( Fig.5-B ).
The metabolic pathways related to nitrogen fixing in the habitat of lithophytic bryophytes after planting on the rock surface are different from those in the original control soil and exposed rock surface, while the nitrogen fixing metabolic pathways in the original control soil and exposed rock surface are more similar (Fig.6).There are also differences among different species of moss, and the nitrogen fixation metabolic pathways of Hyophila involute are more similar to those of Eurohypnum leptothallum.The metabolic pathways of K00265, K00362, K00266, K02575, and K00370 in the habitat after the planting of lithophytic bryophytes on the rock surface were significantly higher than those in the original control soil, while K0195 and K00261 were significantly lower than those in the original control soil. K15577, K15578, and K15576 are significantly higher in exposed rock habitats than in rocky moss colonized rock surfaces and the original control soil.
The phosphorus metabolism pathway in the habitat of lithophytic bryophytes planted on the rock surface was different from that in the original control soil and exposed rock surface. Moreover, there were differences between the lithophytic mosses, Hyophila involute and Didymodon constrictus having more similar soil phosphorus metabolism functions, the original control soil and the exposed rock surface are similar.K00937, K00951, K00873, K03306, and K01507 were significantly higher in the habitat after lithophytic moss was planted on the rock surface than in the original soil control group, while K01524, K01113, K02039, and K00117 were significantly reduced (Fig.7).
The different species of carbon fixation, nitrogen fixation and phosphorus metabolism in rock surface colonized by lithophytic bryophytes
Fig.8 Changes of functional microbial communities in the habitat of lithophytic bryophytes after planting rock surface, A : significantly different species of carbon-fixing microorganisms, B : significantly different species of nitrogen-fixing microorganisms, C : significantly different species of phosphorus metabolism
There were significant differences in the top 15 species of microorganisms related to carbon fixation among the groups.Smaragdicoccus_niigatensis、Gemmatimonadetes_bacterium、Acidimicrobiaceae_bacterium、Thermoleophilia_ bacterium were significantly higher than those of the original control soil, and the richness of Smaragdicoccus_niigatensis was significantly higher than other groups.However, Actinobacteria_bacterium, Acidobacteria _bacterium, Acidimicrobiia _bacterium, Candidatus_Rokubacteria_bacterium, Iamiaceae_bacterium_SCSIO_ 58843 decreased significantly after the bryophytes colonized rock surface ( Fig.8-A ).
The top 15 species of nitrogen-fixing microorganisms were significantly different among the groups. Smaragdicoccus_niigatensis, Gemmatimonadetes_bacterium, Nocardiaceae_bacterium_YC2-7, Microcoleus_sp._PCC_7113 were significantly higher than the original control soil.Among them, the abundance of Smaragdicoccus_niigatensis and Nocardiaceae _bacterium_YC2-7 in Hyophila involute and Eurohypnum leptothallum was higher than that of other treatment groups.However, Actinobacteria_bacterium、Candidatus_Rokubacteria_bacterium、Acidimicrobiia_bacterium、Chloroflexi_bacterium、Deltaproteobacteria_bacterium、 Rubrobacter_taiwanensis、Streptomyces_thermoautotrophicus、Rhizobiales_bacterium 、Streptomyces_lunaelactis decreased significantly compared with the original control soil ( Fig.8-B ).
The top 15 species of microorganisms related to phosphorus metabolism were significantly different among groups.Chloroflexi_bacterium、Smaragdicoccus_niigatensis、unclassified_d__unclassified、Gemmatimonadetes_bacterium、Archangium_gephyra increased significantly after the rock surface was colonized by Lithophytes.The abundance of Smaragdicoccus_niigatensis was higher in the habitats of Eurohypnum leptothallum and Hyophila involute than that in the habitats of Didymodon constrictus. However, Actinobacteria_bacterium、Acidobacteria_bacterium、Acidimicrobiia_bacterium、Acidimicrobiales_bacterium、Deltaproteobacteria_bacterium、Acidimicrobiaceae_bacterium、Candidatus_Rokubacteria_bacterium decreased significantly after the colonization of lithophytic bryophytes (Fig.8-C).
The relationship between different species and soil physical and chemical properties
The correlation between the microorganisms related to carbon fixation, nitrogen fixation,phosphorus metabolism and the contents of nitrogen, phosphorus, potassium, organic carbon, oxalic acid, acetic acid, malic acid and succinic acid in rock surface soil was analyzed. We found that Acidimicrobiia_bacterium, Acidimicrobiaceae_ bacterium and Acidimicrobiales_bacterium in the top 15 microorganisms related to phosphorus were significantly positively correlated with potassium content in soil, and Deltaproteobacteria_bacterium was positively correlated with phosphorus content in soil ( Fig.9-A ).Alphaproteobacteria_bacterium and Solirubrobacterales_bacterium were significantly positively correlated with succinic acid content in soil, and significantly negatively correlated with soil organic carbon. Acidobacteria_bacterium, Solirubrobacterales_bacterium and Acidimicrobiaceae_bacterium were significantly negatively correlated with malic acid in soil. Solirubrobacterales_bacterium and Archangium_gephyra were significantly negatively correlated with acetic acid in soil.
Among the top 15 microorganisms related to carbon fixation, Iamiaceae_ bacterium_SCSIO_58843 was significantly positively correlated with potassium content in soil. Gemmatimonadetes_bacterium was positively correlated with soil organic carbon. Chloroflexi_bacterium was significantly positively correlated with oxalic acid content in soil. Acidimicrobiaceae _ bacterium was significantly negatively correlated with malic acid in soil. Betaproteobacteria_bacterium and Solirubrobacterales_bacterium were significantly positively correlated with succinic acid in soil (Figure 9-B).
Among the top 15 microorganisms related to nitrogen fixation, Candidatus_ Rokubacteria_bacterium_3112 was positively correlated with potassium content in soil.Microcoleus_sp._PCC_7113 was significantly negatively correlated with potassium content in soil. Candidatus _ Rokubacteria _bacterium_3112, Microcoleus _ sp._PCC_7113, Nocardiaceae_bacterium_YC2-7 were positively correlated with soil nitrogen content but not significant.Gemmatimonadetes_bacterium was significantly negatively correlated with oxalic acid.Smaragdicoccus_niigatensis, Gemmatimonadetes_bacterium, Nocardiaceae_bacterium_YC2-7 were significantly negatively correlated with succinic acid in soil. Solirubrobacterales_bacterium was significant positive correlation with succinic acid ( Fig.9-C).