| The largely distributed karst area in China is a representative karst terrain in the world,accounted for 27.9% of China's land area and covers approximately 3.44×106 km~2.The monsoon climate and widely distribution of carbonate rocks,such as dolomite and limestone,make China become one of the countries with the most diverse caves in the world.Caves have abundant records about the evolution of the environment and the earth,and aroused the wide concern by geoscientist and microbiologist.The in-depth knowledge about the interactions between life and environment stems from the diversity and functional research of microbes in extremely environments such as caves.However,for a long time the most cave microbiology studies focused bacterial communities,whilst cave fungi were far less studied.To the best of our knowledge,there are no detailed studies describing the presence and distribution of fungal communities in pristine cave ecosystems in China.Caves are generally considered extreme environments due to the complete darkness and limited nutrients to sustain life.Understanding the fungal communities and their functional groups are of importance in ecology and scientific research.Therefore,Heshang Cave,a pristine solution cave which locates in the south bank of the Qingjiang River in Changyang county,Yichang city,Hubei province,was selected as the study area and samples from the five different niches were collected.We used the microbial ecology methods coupled with mineralogy methods to illustrate the diversity of fungal communities and the functional groups in Heshang Cave ecosystems.The main progresses are as follows.1.A total of 15 pooled samples representing five different habitats in Heshang Cave were sequenced by Illumina HiSeq 2500 platform,resulting in 778 Mb reads and 1,012,715 sequences.Representative sequences of 2,179 OTUs were annotated from phylum to genus level using the default setting of QIIME software and the main taxonomy of them were pertained to 453 genera,72 orders,19 classes and 6 phyla.Ascomycota was the dominant group with the relative abundance of 72.75% and possessed 916 OTUs and 573,274 sequences.We detected 10.66 %,84,614 reads and 653 OTUs for Basidiomycota,8.02 %,63,647 reads and 54 OTUs for Zygomycota,0.14 %,1,081 reads and 32 OTUs for Chytridiomycota,0.09 %,706 reads and15 OTUs for Rozellomycota and 0.06 %,498 reads and11 OTUs for Glomeromycota.At the genus level,the top ten genera were Penicillium(11.21%),Chaetomium(6.88%)Neogymnomyces(5.47%),Aspergillus(2.72%),Mallochia(1.41%),Gymnoascus(1.11 %),Mortierella(5.92%),Mucor(0.94%),Circinella(0.86%)and Geastrum(1.83 %).2.The fungal community richness and community diversity were higher in the dripping water,followed by weathered rocks,sediments,bat guano and air samples.There are significant difference for diversity of fungal communities as evidenced by Chao 1(P < 0.01),ACE(P < 0.01)and Observed species(P < 0.01).Also,dripping water and bat guano samples has significant difference in diversity of fungal communities as evidenced by Shannon(P < 0.01)and Simpson index(P < 0.01).Beta diversity based on the weighted Unifrac analysis showed that there were significant difference between sample of air and weathered rocks(P < 0.01).There were obvious differences in fungal communities between air and the other four niches as evidenced by PCoA and NMDS analysis.We detected 25 biomarkers as the indicate groups and 7 of them possessed higher LDA score(LDA score >5,P < 0.05).Sordariomycetes and Hypocreales were indicator groups of weathered rocks,while Basidiomycetes and Agaricomycetes were indicator groups of dripping waters.We considered Penicillium.mallochii and Ascomycota.sp.to be as indicator groups of air samples and Onygenales as indicator groups of bat guanos.The fungal diversity of air in the innermost part is higher than the entrances.And best of all,based on the deep sequencing data of weathered rocks,the main fungal communities of rocks have been reconsidered.3.We detected 11 classes in coexistence model of the cave fungal communities.Of them,Agaricomycetes has higher betweenness centrality and as the main group in the network relations.Sordariomycetes is widely distributed in the network relations and also connected with other groups.We obtained the 12 guild of the cave fungi after the annotation in the database of FUNGuild.There are three nutritional types for fungi in Heshang Cave and the saprotroph be the dominant.The saprophytic was higher in the bat guanos,followed by the air,dripping water,weathered rock and sediment samples.Pathotroph was higher in air(17.25%)and lower in dripping water samples(4.63%)and plant pathogens as the main groups.Symbiotroph was abundant in dripping water samples(10.48%)and lower in bat guanos(3.11%).Undefined saprophytic fungi and wooden saprophytic fungi were dominated in five niches in Heshang Cave.4.Altogether 194 isolates of indigenous cave fungi were obtained from 42 samples using six types of solid media.The ITS sequences were clustered into 41 OTUs with a cutoff of 5%,which fell into 33 genera within 15 orders of three phyla.46 strains isolated from weathered rocks and belong to the 7 genera in 4 orders of Ascomycota,whilst 85 strains isolated from sediments and belong to the 28 genera in 13 orders of 3 phyla.Only 7 genera in 4 orders of Ascomycota were obtained from 63 strains in bat guanos.Ascomycota was dominated in three niches.Penicillium was dominated genus and accounted for 40%?54% and 52% in sediment,weathered rock and bat guano samples,respectively.Most of the isolates were recovered with MA(24%),CZA(23%),PDA(22%),and SDA(20%)media,indicating the applicability of these media in isolating cave fungi.CFU counts on PDA in the three habitats were the highest in sediments followed by bat guanos and weathered rocks with a value of(3.6±0.001)×103 CFU.g-1,(9.81±0.07)×102 CFU.g-1,and(1.79±0.03)×102 CFU.g-1,respectively.To date,36 genera have been most frequently isolated from solution caves and mines around the world,among them 16 were also isolated in this study from the Heshang Cave.Moreover,we found 23 out of 33 genera in this study were previously reported in carbonate caves.To our knowledge,10 unique fungal genera,such as Metacordyceps,Stilbella,Paraphaeosphaeria,Myriodontium,Auxarthron,Microdiplodia,Trametes,Bjerkandera,Ceriporia and Trichosporon,have yet to be reported in solution caves around the world to date.5.Experiments about fungal strains inducing mineral formation was conducted under laboratory conditions.Results show that the fungi produced oxalic acid accompany with the metabolic activity and formation of crystal with calcium ions.XRD analysis confirmed the formation of calcium oxalate with various morphologies,such as parallelograms,plates,spheres,bipyramids etc.The result indicated that fungi participate in karstification in natural conditions and will eventually affect the stability of carbonates.The innovation of the dissertation is as following.We successfully accessed the completely information for the diversity of cave fungal communities.We revealed indicator groups of fungi in different habitats and confirmed Sordariomycetes as dominate fungal group in rocks,which proposed a new view on the dominated group of rocks.We obtained 10 unique fungal genera which have yet to be reported in solution caves around the world to date. |
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