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This research provides a comprehensive dataset that should be used as a foundation for future, more directed studies, focusing on some of the limitations already discussed in this chapter. It is likely that environmental changes actively select for finer-scale phenotypic or metabolic traits not detectable via the taxonomic analyses (i.e., 16S rRNA gene sequencing) undertaken in this study275. Additionally expanding focus on genome-wide analysis could infer other factors beyond physicochemistry and dispersal limitation that induce both spatial and temporal change in thermophilic taxa, for example, diversification and ecological drift, through tracing gene flow and determining recombination efficiency13,122,346,347.

In all chapters of this thesis, putative metabolic function was assumed from gene-based analysis. Further work is needed to accurately define microbial metabolism and function in these ecosystems as an extended description of population and community dynamics, either in situ in geothermal features through transcriptomics348, in a controlled, chemostat-based approach in the laboratory184, or using 16S rRNA genes or metagenomics to infer predicted function from curated databases349,350. It is important to note that these functional databases are not yet at the same scale as those for taxonomic markers351. Some ecosystem capability through vital and/or novel taxa may go unnoticed if functionality alone is used to define microbial behaviour in any habitat. Determining actual function and cellular state, rather than genetic potential, of geothermal ecosystems, could lead to increased biotechnological application for novel enzymes from extremophiles, which have previously been used in pharmaceuticals, molecular biology, food products, industrial applications, greenhouse gas capture, and bioremediation352,353. Additionally, for Venenivibrio, it would be interesting to ascertain what genes were upregulated in response to certain environmental parameters that might explain niche preferentiality for the taxon, for example, by using transcriptomics to measure phenotypic response during arsenic stress354 or resolve the uptake of sulfur into the cell355. Also, the full high affinity molybdate uptake system (modABCD) was not annotated in the V. stagnispumantis CP.B2T genome, whereas the operon was present in all other Hydrogenothermaceae genomes analysed. This could expand on the apparent lack of

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mixotrophy within Venenivibrio, or give Sulfurihydrogenibium a competitive advantage in colonising a habitat amenable to both taxa, as molybdenum is an essential co-factor in molybdoenzymes used for bacterial metabolism (e.g., formate and sulfite dehydrogenases)356. CP.B2T also had fewer copper exporting ATPases (copB), again compared to other family members, so using a function-based assessment like transcriptomics could narrow defining characteristics that have enabled the apparent endemism of the genus to Aotearoa-New Zealand.

Further, this thesis focused only on prokaryotic (i.e., archaeal and bacterial) communities and populations. Geothermal microbial ecosystems also include eukaryotic microorganisms, such as microalgae148 and fungi357, as well as viruses358, so targeted research on these taxa would complement findings presented through this study. Indeed, the data generated by Chapter 2 has already been utilised to describe protistan diversity in TVZ geothermal springs156, adding to a vital, but under-researched, component of ecosystem services in these habitats. As already stated in this chapter, the original Earth Microbiome Project primers (515f-806r) used by this study are known to contain biases towards Bacteria, and so, could have potentially eliminated detection of many Archaea in these ecosystems, in particular Thermoproteota and Nitrososphaerota232. It would be prudent for future study, in particular for geothermal sediments which are known to harbour increased abundance of Archaea172, to use the modified 515f-806rB primer pair that are highly concordant with the original primer set to allow cross-comparison between studies232. An additional archaeal-specific primer set could also be used, if the focus was on identifying rare taxa (and associated abundances) underrepresented by the current study359, which could also be mixed into one reaction mix with universal bacterial primers to ensure no partiality for either domain. Particularly, these primers could be used in conjunction with digital PCR to obtain absolute taxa abundances360. The collection and cryogenic storage of associated sediments from each geothermal spring water column collected for this study, along with the water column DNA already extracted and archived, will provide a unique opportunity to assess these shortcomings361. Ideally, using both universal and archaeal-specific primers would give the best comparison to the current dataset and representation of realistic archaeal diversity in TVZ geothermal springs.

Metagenomic sequencing would also remove the biases of any primer set, but as sequencing and analysing metagenomes from >1,000 sites is not likely attainable for the immediate study of Aotearoa-New Zealand geothermal systems and would reduce biodiversity resolution of target assemblages, complementing 16S rRNA gene data with a reduced number of sample

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metagenomes would aid confidence in defining microbial ecosystem behaviour in these environments.

While the focus of Chapter 4 was on the most abundant genus found in TVZ geothermal water columns, assessment of other populations, preferably both abundant and rare, remains to be examined. We know that some extremophiles were missed by the study design, for example, the novel genus and species from the Thermoproteota phylum, Zestosphaera tikiterensis, and its symbiotic nanoarchaeote, Candidatus Nanoclepta minutus. Both were recently isolated from anaerobic enrichment cultures taken from the Tikitere geothermal field in the TVZ362. Even though some taxa have either evaded detection or have not appeared in the broad-range analysis from this thesis, the dataset can be bioprospected to find specific geothermal niches with the appropriate physicochemical environment conducive to growth of a target microorganism. Enrichment of samples from geothermal springs with increased 16S rRNA gene abundance of the desired taxon can also aid in isolating pure cultures, which has already been utilised in the recent isolation of six divergent strains of Venenivibrio spp. from the TVZ (unpublished data). Obtaining and characterising novel isolates, or isolating co- cultures, is still imperative to determine microbial phenotypes and physiology, improve gene- based annotations, discover innovative biotechnological applications, decipher complex biotic interactions, and eliminate relic DNA as a contributor to community structures337,363–

365. Additionally, elucidating drivers of the abundant acidophile Acidithiobacillus in an alternate physicochemical niche to Venenivibrio, in conjunction with the genome-wide analysis of the taxon in TVZ sinters and sediments9, would enhance understanding of extremophilic biogeography in the region. Taxa from the phylum Armatimonadota were also found to be widespread in TVZ geothermal springs195, but this novel phylum, first identified in 1998158 and characterised in 2011366, remains under-explored and -described367. The diversity and biogeography of Armatimonadota could be analysed in detail using this dataset, which could then inform future research on the metabolism, physiology, and ecosystem function of this novel phylum. With the exception of Venenivibrio, this thesis primarily focused on large-scale community patterns of diversity to gain initial insight into these novel ecosystems, providing a fundamental infrastructure for future, more targeted research on individual populations and taxa.

Indeed, population analysis via the genus Venenivibrio has introduced many questions. The apparent endemism of a bacterial genus is unprecedented. Venenivibrio presents an easily

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accessible and tractable genus to validate this finding, as the overwhelming presence and abundance in Aotearoa-New Zealand geothermal springs preclude under-sampling and/or rarity as rebuttals for endemic classification. While allopatry has clearly played a part in the evolution of the genus within the Hydrogenothermaceae family, it is unlikely to be the sole cause of contemporary endemism. Other constraining factors, like physical dispersal mechanisms and environmental stress responses, need to be explored in more detail to assess key metabolic and physiological traits that either drive and/or maintain the apparent endemism. Multiple modes of transport could be investigated (i.e., aerosols, spring drainage streams, and/or geothermal aquifers), with the absence of Venenivibrio internationally likely reflecting a strong negative selection pressure (via pH, salt, and starvation extremes) in long- range atmospheric transport327. These barriers to migration are further enhanced by a lack of suitable environmental stepping stones immediately beyond Aotearoa-New Zealand, and this aspect could be more conclusively examined by including the nearest geothermal springs outside of the archipelago (e.g., in New Caledonia or Tonga)368,369 for future research on the genus. Indeed, traces of the taxon were found in geothermal features on Raoul Island, as highlighted in Table D.2, Appendix D370. This island, situated ~1,100 km northeast of the North Island, is the northernmost outpost of Aotearoa-New Zealand and reinforces the concept that local dispersal of Venenivibrio is not restricted. Results from this chapter also show that while narrow pH and temperature optima increase abundance of Venenivibrio in specific spring environments, tolerance of broader environmental conditions do exist within the genus, signifying the importance of future investigation at species- and strain-level.

Additionally, the timeline of diversification within the Hydrogenothermaceae needs to be validly elucidated, with preliminary molecular dating suggesting divergence between Venenivibrio and Sulfurihydrogenibium occurred ~67 mya318. Most significantly, deciphering how other family members have successfully managed intercontinental dispersal, along with the dispersal-limiting mechanisms of Venenivibrio preventing dissemination onto the global stage, will be essential to explaining the isolated ubiquity of the genus in the Aotearoa-New Zealand archipelago. Investigating the diffusivity (i.e., a measure of dispersal rate over evolutionary time scales)109 of the taxon would complement this avenue of exploration, as would more extensive comparative genomics of the Hydrogenothermaceae to differentiate metabolic capability between the genera. Elucidating the role of local historic events (i.e., the separation of Zealandia from the Gondwana supercontinent via seafloor spreading and the Oruanui supereruption of the Taupō volcano)333,334, combined with the legacy of other

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Hydrogenothermaceae79, will not only further establish the concept of endemism for Venenivibrio, but also enhance our understanding of microbial evolutionary processes.

Finally, the data and findings produced by this thesis are available to researchers and bioinformaticians to support model development of microbial community establishment and structure, ecosystem health, and uniqueness119,371–373. The study provides sufficient data to robustly approximate community assembly using typical taxon-geochemical associations and geographical influence. The research also complements microbial ecology studies of non- extreme biomes in Aotearoa-New Zealand374–377, and increases knowledge on the value of indigenous biodiversity in the country. Assessing ecosystem health and the effect of environmental change in geothermal springs is complex, in comparison to non-extreme environments227,371, as the range of physicochemistry and microbial taxa found in these habitats make establishing a baseline difficult. This study provides a starting point, however, to determine typical microbial assemblages and corresponding physicochemical types378, in particular to specific geothermal fields, that could proxy for environmental health and resilience in these novel habitats.

This study clearly provides a springboard to assess the cultural, recreational, and resource development value of the microbial component of geothermal springs, both in Aotearoa-New Zealand and globally. Not only are DNA sequences and associated metadata available to the scientific community in INSDC databases, the microbiological, physicochemical and locational information of each geothermal feature is presented in a user-friendly website (https://1000springs.org.nz/), increasing the scope of the study to several stakeholders. These include the general public in expanding science outreach, landowners and tourism operators of geothermal sites, commercial partners, geothermal power industry, government agencies, and Māori groups (under mana whenua). The findings of each chapter reiterate that microorganisms are taonga (i.e., valuable natural resources), with many of the features included in this research occurring on culturally important and protected land for Māori.

Therefore, this or follow-on future projects provide an avenue for exploration of indigenous knowledge, while assisting in management, conservation, and/or protection effects of national government agencies to maintain these ecosystems as part of Aotearoa-New Zealand’s treasured endemic species.

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