Doctoral Dissertation

Bacterial microbiomes associated with rock-dwelling lichens(Summary)

Author: Zichen HE
Supervisor: Takeshi NAGANUMA

Program of Food and AgriLife Science
Graduate School of Integrated Sciences for Life
Hiroshima UniversityMarch 2023Ꮫ ఩ ㄽ ᩥ ࡢ せ ⣙  ㄽᩥ㢟┠   Bacterial microbiomes associated with rock-dwelling lichens(ᒾ▼╔⏕ᛶᆅ⾰㢮࡟ඹᅾࡍࡿ⣽⳦┦࡟㛵ࡍࡿ◊✲)                   ᗈᓥ኱Ꮫ኱Ꮫ㝔⤫ྜ⏕࿨⛉Ꮫ◊✲⛉                        㣗ရ⏕࿨⛉Ꮫࣉࣟࢢ࣒ࣛ                   Ꮫ⏕␒ྕ 㹂㸰㸮㸴㸱㸯㸰                   Ặ  ྡ ఱ Ꮚ⌵ (HE Zichen)
Lichens are mutually symbiotic systems consisting of fungal and algal symbionts.
While diverse lichen-forming fungal species are known, limited species of algae form
lichens. Plasticity in the combination of fungal and algal species with different ecophysiological properties may contribute to the worldwide distribution of lichens, even in
extreme habitats. Lichens have been studied systematically for more than 200 years;however, plasticity in fungal–algal/cyanobacterial symbiotic combinations are still
unclear. In addition, the association between non-cyanobacterial bacteria and lichens has
attracted attention in recent years. The types, diversity, and functions of lichen-associatedbacteria have been studied using both culture-based and culture-independent methods. In
this study, Rock tripe lichens (Umbilicariaceae) were collected from Continental andMaritime Antarctic biological, Arctic and Subarctic Areas in North America and NorthernEurope, Alpine Areas in Eastern Alps and Equatorial Africa and Japanese regions; NonUmbilicaria lichens were collected from Guiana and South Africa regions; in order tocompare their bacterial floras and potential metabolism. Bulk DNA extracted from thelichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S
rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The dataof each sample were analyzed by regions, and the results of Umbilicaria and NonUmbilicaria lichens were comparing respectively. Finally, the results of the overall rockdwelling lichens were integrated and analyzed.
This doctoral dissertation consists of seven chapters: Chapter 1. Introduction:
Chronicle of Research into Lichen-associated Bacteria; Chapter 2. Microbiomic Analysisof Bacteria Associated with Rock Tripe Lichens in Continental and Maritime AntarcticRegions; Chapter 3. Bacterial Microbiomes Associated with Rock Tripe Lichens from
Arctic/Subarctic Areas in North America and Northern Europe; Chapter 4. Microbiome
Analysis of Bacteria Associated with Rock Tripe Lichens from Alpine Areas in Eastern
Alps and Equatorial Africa; Chpater 5. Microbiomes associated with rock tripe lichensdwelling on montane rocks and cliffs in western Japan; Chpater 6. Microbiomes
Associated with Epilithic Non-Umbilicaria Lichens from the Venezuelan Guiana Shield
and the South African Highveld Plateau; Chpater 7. General discussion of approximate
global rock-dwelling lichens.
Chapter 1. Introduction: Chronicle of Research into Lichen-associated Bacteria
reviewed the research history of lichens, including the researches in the past more than
200 years and lichen-associated bacteria and provides insights into the current status ofresearch in this field. As to bacterial associates of lichens, traditional culture-based
methods reveal their physiological and biochemical features in vitro but allow onlyinferences on their roles in vivo in lichens. Culture-independent molecular approaches
provide taxonomic and phylogenetic identifications of bacterial associates but only insilico speculations on their functions in lichens. Considering the variety and practicality

of secondary metabolites produced by lichen-forming fungi and bacteria, lichenassociated bacteria would represent huge treasure houses for human benefit. However,research on lichen-associated bacteria is still limited.
Chapter 2. Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens inContinental and Maritime Antarctic Regions collected rock tripe lichens from two distinct
Antarctic biological regions, the continental region near the Japanese Antarctic station(Syowa Station) and the maritime Antarctic South Orkney Islands (Signy Island), in order
to compare their bacterial floras and potential metabolism. Bulk DNA extracted from thelichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S
rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The
fungal and algal partners represented members of the ascomycete genus Umbilicaria andthe green algal genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4
sequences were grouped into operational taxonomic units (OTUs), which were assigned
to eight bacterial phyla, Acidobacteriota, Actinomyceota, Armatimonadota, Bacteroidota,
Cyanobacteria,

Deinococcota,

Pseudomonadota

andthecandidate

phylumSaccharibacteria (also known as TM7), commonly present in all samples. The OTU
floras of the two biological regions were clearly dis-tinct, with regional biomarker genera,such as Mucilaginibacter and Gluconacetobacter, respectively. The OTU-basedmetabolism analysis predicted higher membrane transport activities in the maritime
Antarctic OTUs, probably influenced by the sampling area’s warmer maritime climaticsetting.
Chapter 3. Bacterial Microbiomes Associated with Rock Tripe Lichens from
Arctic/Subarctic Areas in North America and Northern Europe collected rock tripe lichensfrom Arctic and Subarctic biological regions, in order to compare their bacterial floras

and potential metabolism. Bulk DNA extracted from the lichen samples was used toamplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA gene, whoseamplicons were Sanger- and MiSeq-sequenced, respectively. The fungal and algal
partners represented members of the ascomycete genus Umbilicaria and the green algal
genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4 sequences weregrouped into operational taxonomic units (OTUs), which were assigned to ten bacterial
phyla, Acidobacteriota, Actinomycota (formerly Actinobacteria), Armatimonadota,
Bacteroidota(formerlyBacteroidetes),

Chloroflexota,

Cyanobacteria,

Gemmatimonadota, Planctomycetota, Pseudomonadota (formerly Proteobacteria) andVerrucomicrobiota, commonly present in all samples. The OTU floras of the twobiological regions were clearly distinct, with regional biomarker genera, such as
Mucilaginibacter and Gluconacetobacter, respectively. Even though the lichen samples
with various lichen species, the bacterial OTUs were clustered on the dendrogram
between different sampling regions Arctic and Subarctic. This chapter think that thegeographical and/or bioclimatic environment, rather than the different lichen-forming
fungus species, is what affects the bacterial microbiomes linked to the Umbilicaria
species investigated here, And the climate change of sampling sites may actually havesimilarities.
Chapter 4. Microbiome Analysis of Bacteria Associated with Rock Tripe Lichens fromAlpine Areas in Eastern Alps and Equatorial Africa collected rock tripe lichens from two
alpine fellfields of Mt. Brennkogel in Eastern Alps (Austria) and Mt. Stanley of theRwenzori mountains in equatorial Africa (Uganda), to compare their bacterialcompositions, diversities, possible biomarkers and potential metabolisms. Bulk genomic
DNA was extracted from the lichen samples, which were used to amplify the 18S rRNA

gene by Sanger sequencing and the V3-V4 region of the 16S rRNA gene by Illumina
Miseq sequencing. Based on 18S rRNA gene sequences, the symbiotic components offungal and algal partners were represented by the genus Umbilicaria belonging toAscomycota and the green algal linegae Trebouxia belonging to Chlorophyta, respectively.
The reads of V3-V4 region were classified into operational taxonomic units (OTUs)
according to a set value of similarity, which were assigned to a total of 26 bacterial phyla,
were all found in both areas. Eight of the 26 phyla, i.e., Acidobacteriota, Actinomycota(formerly Actinobacteria), Armatimonadota, Bacteroidota, Chloroflexota, Deinococcota,
Planctomycetota, and Pseudomonadota (formerly Proteobacteria), were present in all
samples with read abundances of >1% of the total read number. The bacterial
compositions of the Austrian and Ugandan lichens were distinct, with the OTU
frequency-based regional biomarker phyla, Pseudomonadota and Armatimonadota,
respectively. The OTU-based potential metabolism analysis predicted similar relative
abundances of metabolic pathways in the two regions, probably influenced by similar
alpine climate, although the distance between the two areas is calculated as about 5430
km.
Chpater 5. Microbiomes associated with rock tripe lichens dwelling on montane rocks
and cliffs in western Japan collected rock tripe lichens from Japan, in order to compare
their bacterial floras and potential metabolism. Bulk DNA extracted from the lichen
samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA
gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The fungal andalgal partners represented members of the ascomycete genus Umbilicaria including twospecies U. esculenta and U. muehlenbergii, and the green algal genus Trebouxia, basedon 18S rRNA gene sequences. The V3-V4 sequences were grouped into operational

taxonomic units (OTUs), which were assigned to ten bacterial phyla, Acidobacteriota,
Actinomycota (previously Actinobacteria), Bacterroidota (formerly Bacteroidetes),
Chloroflexota, Cyanobacteria, Gemmatimonadota, Planctomycetota, Pseudomonadota(before Proteobacteria), and Saccharibacteria_TM7. Although it is grouped according to
two different species, in fact, the difference of bacterial communities in the clothing
samples in the sampling area has nothing to do with species. That is, the associatedbacterial communities are almost no-grouping, and the results were not show variatedlichen species-specific grouping.
Chpater 6. Microbiomes Associated with Epilithic Non-Umbilicaria Lichens from theVenezuelan Guiana Shield and the South African Highveld Plateau collected epilithic
non-Umbilicaria lichens from the Venezuelan Chiuri tepui and the South African
Highveld Plateau, in order to compare their bacterial floras and potential metabolism.
Bulk DNA extracted from the lichen samples was used to amplify the 18S rRNA geneand the V3-V4 region of the 16S rRNA gene, whose amplicons were Sanger- and MiSeqsequenced, respectively. The fungal and algal partners represented members of thecommon lichens, including fruticose and foliose, and the green algal genus Trebouxia,
based on 18S rRNA gene sequences. The V3-V4 sequences were grouped into operationaltaxonomic units (OTUs), which were assigned to twelve bacterial phyla, Acidobacteriota,Actinomycota (formerly Actinobacteria), Armatimonadota, Bacteroidota (formerlyBacteroidetes), Chloroflexota, Cyanobacteria, Deinococcota, Gemmatimonadota,
Planctomycetota, Pseudomonadota (formerly Proteobacteria), Saccharibacteria_TM7and Verrucomicrobiota. Although lichens collected in these two regions are different, themicrobial communities in these two regions have obvious regional characteristics
especially at Class, Genus and Species ranks. On the other hand, in the same samplingregion, lichen samples of the same species or genus tended to cluster. This discovery
might provide evidence for the influence of local environment and lichen species onlichen associated bacterial community, and the influence of local environment is greaterthan lichen species.
Chpater 7. General discussion of approximate global rock-dwelling lichens contained
simple discussion of fungal and algal partners a total of 82 lichen samples; and mainly
focused on and analyzed the V3-V4 MiSeq data of a total of 82 lichen samples in theabove chapters to seek global overall information of rock-dwelling lichens associatedbacterial microbiome. The analyses of bacterial microbiomes communities determinedthe detailed biogeographic information in different regions by bioinformatics, especially
the Syowa Station in Antarctica showed a clear boundary with other regions regardless oflichen types. Roles or functions of different bacterial microbiomes communities werepredicted according to biomarkers, however, no matter how the bacterial microbiome oflichens is, the overall structure of their metabolic pathway prediction results will notchange significantly. In addition, cosmopolitan and potential cosmopolitans were foundand introduced.