A foundational biodiversity map of the terrestrial microbiome of a sub-Antarctic island.

Siddarthan Venkatachalam1, Robert Pienaar1, Gwynneth Matcher1, Nasipi Mtsi1, Peter le Roux2, Michelle Greve2, Bettine Jansen van Vuuren3, Sudharshan Sekar3, Rosemary Dorrington1

1Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 2Department of Plant and Soil Sciences, University of Pretoria 3Department of Zoology, University of Johannesburg Sub-Antarctic Island: Prince Edward Islands

 Marion Island (290 km2) and Prince Edward (45 km2)1  PEIs are of volcanic origin  Important breeding site for seabirds and mammals2  Gradual increase 0.04°C per year  Decrease in annual precipitation  Fragile ecosystem

(courtesy of I. Meiklejohn).

Marion Island (46° 54´ S, 37° 45´ E) Prince Edward Island (46° 38´ S, 37° Marion Island Vegetation

The vascular flora (N.J.M. Gremmen, pers. comm.)  22 native species,  18 introduced species  3 unknown species

Indigenous plant

Azorella selago Hook. f.

Invasive plant

Sagina procumbens L.

Courtesy of Robert Pienaar Over all objective

To map the genetic diversity of microbial communities and plant species present in the Marion Island under different habitat complexes.

Research Questions

1. Does different habitat complexes in the Marion island contains different microbial communities? 2. What microbial groups are associated with the root tissues of plants (rhizosphere), surrounding soils and how do plants benefit from their microbiome? 3. Do different soils and plants types host different microbial communities? 4. Is there any significant differences among rhizosphere microbial communities of indigenous Azorella selago and invasive Sagina procumbens plant species? If so how they are influencing the sub-Antarctic ecosystems.

Sample collection under different habitats

Rachomytrium Habitat Soil Azorella Blechnum Sagina Crassula Cotula Agrostis Acaena (moss) Fellfield 8 7 2 1 Nil Nil Nil Nil 1 Polar 3 Nil Nil Nil Nil Nil Nil Nil Nil desert Salt spray 2 6 Nil 9 8 6 Nil Nil Nil Mire-Slope 8 2 6 8 Nil Nil 1 1 Nil

Samples were collected during 2016 Marion takeover voyage, (April/May) Molecular Taxonomy

Bacteria : 16S rRNA gene Eukaryotes : 18S rRNA gene

E517F (5’-CAGCAGCCGCGGTAA-3’) E969-984 (5’-GTAAGGTTCYTCGCGT-3’) Highly conserved regions Plant Barcoding genes – Identical in all bacteria – Single PCR primer pair can amplify 16S rRNA  Ribulose biphosphate genes from diverse carboxylase large (rbcL) bacteria Highly variable regions  Maturase K (matK) – Conserved within species – Divergent between species

Plant Barcoding

Vascular indigenous plant species barcoded in Marion Island

Acaena magellanica, Agrostis castellana, Agrostis magellanica, Blechnum penna-marina, Callitriche, Cotula plumose, Crassula moschata, Elaphaglossum, Grammitis kerguelensis, Hymenophyllum peltatum, Juncus, Limosella, https://wikivisually.com/lang-es/wiki/Acaena_magellanica Lycopodium magellanicum, Lycopodium saururus, Montia fontaum, Poa cookii, Polystichum, Pringlea antiscorbutica, Ranunculus biternatus, Uncinia compacta. Microbial community diversity between different habitats Phylogenetic classification

Phylogenetic classification of bacterial reads represented by heat map. The bacterial 16S rRNA gene sequences were amplified from gDNA and classification was carried out using the Silva reference database (Version 128) and the Mothur software (Version 1.39) platform. Phylogenetic classification

Phylogenetic classification of bacterial reads represented by heat map. The bacterial 16S rRNA gene sequences were amplified from gDNA and classification was carried out using the Silva reference database (Version 128) and the Mothur software (Version 1.39) platform. Non-Metric Multidimensional Scaling Plot

Non-metric multidimensional scaling (nMDS) ordination plots. The 16S rRNA amplicon libraries generated from gDNA. The 2D-NMDS was standardized by square-root transformation and a Bray–Curtis dissimilarity matrix. The 16S rRNA bacterial OTUs generated (cut-off 97%) Microbial community diversity between indigenous and invasive plant Azorella selago Hook. f.  One of the primary colonizers3  Most abundant and widely distributed4  High impact on Marion Island ecosystems  Ecosystem engineers involves in soil formations  Provides microclimates for microbes and small insects

Sagina Procumbens L. Greve et al. 2017

 First found on Marion Island in 19655  Increasing distribution on Marion6  Can colonize new environments (e.g. Surtsey)7

Diagram of sampling Indigenous and Invasive plant 12 Exposed Soil

Soil Underneath 9 Cushion 3

Soil Control 6 Phylogenetic classification

100% Acidobacteria Actinobacteria 90% Bacteroidetes Chlamydiae 80% Chloroflexi

70% Cyanobacteria Dependentiae 60% Fibrobacteres Firmicutes 50% Gemmatimonadetes Latescibacteria 40% Minor Phyla

30% Nitrospirae Patescibacteria Abundance Relative to Sample Total (%) Total Sample to Relative Abundance 20% Planctomycetes Proteobacteria 10% Rokubacteria Spirochaetes 0%

M1 M3 Unclassified Bacteria M10 M12 M19 M21 M27 M29 M35 M37 M43 M45 M52 M54 M60 M62 RR082 RR085 RR092 RR100 RR102 RR109 RR111 RR119 RR121 RR128 RR130 RR137 RR139 RR146 RR148

RR084r1 RR093r2 Verrucomicrobia ROS 1 ROS 2 ROS 3 ROS 4 ROS 5 ROS 6 ROS 7 ROS 8 SAG 1 SAG 2 SAG3 SAG 4 SAG 5 SAG 6 SAG 7 SAG 8

Soil Sample Underneath Plant Body Principal Coordinates Analysis

Fast UniFrac based PCoA ordination plots. The 16S rRNA amplicon libraries generated from gDNA and The 16S rRNA bacterial OTUs generated at the cut-off 97%. The samples were standardized by square-root transformation and a Bray– Curtis dissimilarity matrix. Summary

 Diversity and distribution of bacterial communities across different habitat complexes in Marion Island were studied.  Saltspray habitats contains distinct bacterial diversity pattern  Bacterial assemblages between indigenous Azorella selago and invasive Sagina procumbens found to be significantly different.

Future Work  Priority habitat sites-  Functional Genomics: Metabolic profiles of bacterial genomes

Other flagship programs

Marine spatial planning: Algoa Bay R.A. Dorrington and A.T. Lombard

Antarctic Circumnavigation Expedition Sarah Fawcett and R.A. Dorrington Other flagship programs

Stromatolites biodiversity survey: Gordon and Betty Moore foundation - R.A. Dorrington and Kerry L Mcphail

Freshwater Lake : Algal Blooms - Guy Bate and R.A. Dorrington References

1 Venkatachalam S. et al., 2017. PloS One. 12 (8), e0183400 2Venkatachalam S. et al., 2018. Limnology and Oceanography online first 3McGeoch, M. A. et al., 2008. Stellenbosch, RSA: Sun Press, pp. 215–276. 4Phiri, E.E., McGeoch, M.A. & Chown, S.L. 2015. Polar Biology. 38(11):1881– 1890 5Gremmen, N. 1975. South African Journal of Antarctic Research. 5:25–30. 6Ramaswiela, T. 2010. MSc thesis. Stellenbosch University. 7Magnússon, B. et al. 2009. Surtsey Research. 12(5):57–76.

ACKNOWLEDGEMENTS

NRF SARchi

Marine Natural Products Lab, 417 Any Queries /Collaboration /Assistance in genomics platform

Dr. Venkat Siddarthan, Post-Doctoral Researcher, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown Email: [email protected]

Bacterial Communities – Azorella selago Azorella 1 - Large Azorella 3 - Large

Azorella 5 - Large Azorella 7 - Large Bacterial Communities – Azorella selago Azorella 2 - Small Azorella 4 - Small

Azorella 6 - Small Azorella 8 - Small Bacterial Communities – Sagina procumbens

Sagina 1 - Large Sagina 3 - Large

Sagina 5 - Large Sagina 7 - Large Bacterial Communities – Sagina procumbens Sagina 2 - Medium Sagina 8 - Medium

Sagina 4 - Small Sagina 6 - Small