The Koala (Phascolarctos cinereus) faecal microbiome differs with diet in a wild population

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Microbiology

Main article text

 

Introduction

Background—Cape Otway

Experimental Methods

Study species

Faecal sample collection

Analysis of bacterial community through rRNA gene sequencing and analysis

Cyanobacteria identity and chloroplast contamination assessment

Leaf collection method

Diet composition

Diet quality

Comparison of potential functional differences between Parabacteroides and Ruminococcaceae genomes

Results

Bacterial community rRNA gene sequencing

Functional differences between previously identified and publicly available Parabacteroides and Ruminococcaceae genomes

Diet composition

Diet quality

Discussion

Conclusion

Supplemental Information

Supplementary information methods and results file.

DOI: 10.7717/peerj.6534/supp-1

Google Earth map of Cape Otway 2013 koala collection

Map showing locations of the Cape Otway 2013 koalas located in E. viminalis and E. obliqua trees, within discreet areas of E. viminalis and E. obliqua forest patches. GPS locations were taken at collection during 2013, Google Earth map produced 10th November 2018 (Map data ©2018 Google).

DOI: 10.7717/peerj.6534/supp-2

Google Earth map of Cape Otway 2015 koala collection

Map showing locations of the Cape Otway 2015 koalas located in E. viminalis and E. obliqua trees, within discreet areas of E. viminalis and E. obliqua forest patches. GPS locations were taken at collection during 2015. The GPS coordinates for the koala identified as K13 from the 2015 collection were not recorded, and therefore it is not shown on the map. Its faeces were sampled in the same area as the other E. viminalis koalas. Google Earth map produced 10th November 2018 (Map data ©2018 Google).

DOI: 10.7717/peerj.6534/supp-3

β diversity principal coordinates analysis (PCoA) based on Bray–Curtis for the combined Cape Otway bacterial T-RFLP

Scatterplot from the Bray–Curtis metric for the combined Cape Otway koala population with diets comprising E. viminalis and E. obliqua. Assessment of the Bray–Curtis metric using PERMANOVA indicated no influence of collection year (Pseudo F2 = 1.19 PERMANOVA P = 0.49), although it detected a significant influence of diet (Pseudo F2 = 11.36, PERMANOVA P = 0.0001).

DOI: 10.7717/peerj.6534/supp-4

PCoA of β diversity based on Bray–Curtis metric of bacterial TRFs

Scatterplots of Bray–Curtis bacterial TRFs demonstrated significant separation along the (A) PCoA 1 axis for both the 2013 (Pseudo F1 = 11.75, PERMANOVA P = 0.0001) and (B) 2015 (Pseudo F1 = 5.13, PERMANOVA P = 0.0001) koala faecal samples. SE bars are representative of combined technical replicates.

DOI: 10.7717/peerj.6534/supp-5

PCoA of β diversity based on UniFrac matrices for genus-level OTUs

Scatterplots of weighted UniFrac genus-level OTUs from the Cape Otway (A) 2013 and (B) 2015 koala collection years. PERMANOVA analysis of genus-level OTUs revealed a significant separation between koala diets in (A) 2013 (Pseudo F1 = 56.08, PERMANOVA P = 0.001) and (B) 2015 (Pseudo F1 = 17.04, PERMANOVA P = 0.001).

DOI: 10.7717/peerj.6534/supp-6

PCoAs of β diversity based on Bray–Curtis metric of Archaea and Fungi TRFs for the 2013 koala population

Scatterplots of Bray–Curtis of archaeal 16S and fungal ITS rRNA genes indicated a slight separation along the PCoA axis one for (A) archaea (Pseudo F1 = 4.82, PERMANOVA P = 0.0001. No separation was identified visually by either axis one or two for (B) fungal samples (Pseudo F1 = 2.41, PERMANOVA P = 0.004. SE bars are representative of combined technical replicates.

DOI: 10.7717/peerj.6534/supp-7

Potential functional capacities of Parabacteroides and Ruminococcaceae species genomes

Gene families involved in the metabolism and degradation of plant components i.e., cellulose and the plant secondary metabolite (PSMs) i.e., tannins.

DOI: 10.7717/peerj.6534/supp-8

Parabacteroides phylogenetic tree

The Parabacteroides phylogenetic tree indicates that the 16S rRNA gene sequences isolated from the koala gut microbiomes are dispersed throughout the Parabacteroides tree. The aLRT statistical test for branches values are indicated on each branch.

DOI: 10.7717/peerj.6534/supp-9

Ruminococcaceae phylogenetic tree

The Ruminococcaceae phylogenetic tree indicates that the 16S rRNA gene sequences isolated from the koala gut microbiomes are dispersed throughout the Ruminococcaceae tree. The aLRT statistical test for branches values are indicated on each branch.

DOI: 10.7717/peerj.6534/supp-10

Sequencing statistics for 2013 and 2015 the Cape Otway koala samples

Statistics are provided for the combined Cape Otway population sequences processed through QIIME together after split_libraries.py command to produce the seqs.fna file and then after identify_chimeric_seqs.py commands. The sequences were then split into the 2013 and 2015 collections to be analysed separately for OTUs and taxonomic differences and similarities.

DOI: 10.7717/peerj.6534/supp-11

Alpha diversity for Cape Otway 2013 and 2015 individual koalas

Alpha diversity indices obtained through QIIME “alpha_diversity.py” command. Diversity indices were then analysed per koala. The National Center for Biotechnology Information, Sequence Read Archive (NCBI SRA) accession number assigned to sequences from each koala are provided.

DOI: 10.7717/peerj.6534/supp-12

Alpha diversity for Cape Otway 2013 and 2015 based on diet eaten

Alpha diversity indices obtained through QIIME “alpha_diversity.py” command. Diversity indices were then analysed per diet.

DOI: 10.7717/peerj.6534/supp-13

Alpha diversity for Cape Otway 2013 and 2015 collection year

Alpha diversity indices obtained through QIIME “alpha_diversity.py” command. Diversity indices were then analysed per collection year.

DOI: 10.7717/peerj.6534/supp-14

Relative abundance of genus-level OTUs from 2013 and 2015 koalas eating E. viminalis and E. obliqua

The top 22 bacteria identified to genus level from Cape Otway koalas in eating E. viminalis and E. obliqua in 2013 and 2015. A relative abundance per eucalypt species and total relative abundance for each population is provided. Data obtained from rarefied and summarized genus level OTU tables generated through QIIME. Mean abundance ± 1 SE.

DOI: 10.7717/peerj.6534/supp-15

Family-level comparison of gut microbiome from 2013 and 2015 koalas eating E. viminalis and E. obliqua

Identification of the top 24 bacterial families identified by “summarise_taxa.py” OTUs command in QIIME for Cape Otway koalas in 2013 and 2015. Relative abundance is provided by diet, data obtained from rarefied and summarized family level OTU tables generated through QIIME. Mean abundance ±1 SE.

DOI: 10.7717/peerj.6534/supp-16

Functional potential of Parabacteroides and Ruminococcaceae genomes

Complete or draft linear genomes (at least 30 × coverage) of 35 bacteria from Parabacteroides and 45 from Ruminococcaceae. The genomes and their information were sourced from the Joint Genomes Institute (JGI) through the Integrated Microbial Genomes and Microbiomes (IMG) portal. The website is available through https://img.jgi.doe.gov/cgi-bin/mer/main.cgi.

DOI: 10.7717/peerj.6534/supp-17

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Kylie L. Brice conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Pankaj Trivedi conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Thomas C. Jeffries and Christopher Mitchell analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Michaela D.J. Blyton analyzed the data, authored or reviewed drafts of the paper, approved the final draft.

Brajesh K. Singh contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Ben D. Moore conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

Data Availability

The following information was supplied regarding data availability:

BioProject: PRJNA521666.

Funding

This research was supported under the Australian Research Council’s Linkage Projects funding scheme (project number LP140100751); an Australian Postgraduate Award to Kylie Brice; the Western Sydney Postgraduate top up award and a Paddy Pallin Science Grant Award from the Royal Zoological Society of NSW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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