Chanenath Sriaporn

Kathleen A Campbell

Maeva Millan

Steven W Ruff

Martin J Van Kranendonk

Kim M Handley
email: kim.handley@auckland.ac.nz

Stromatolitic digitate sinters form under wide-ranging physicochemical conditions with diverse hot spring microbial communities

Keywords: biosignature, digitate sinter, stromatolite, hot springs, microbial communities, silicification

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Introduction

In 2007, the Mars Exploration Rover 'Spirit' identified outcrops and sediments composed of opaline silica adjacent to a volcaniclastic landform dubbed Home Plate in the Columbia Hills of Gusev crater [START_REF] Squyres | Detection of silica-rich deposits on Mars[END_REF]. With abundant indications of ancient hydrothermal activity in this area, the opaline silica was inferred to be a hydrothermal deposit produced from fumaroles or hot springs [START_REF] Ruff | Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile[END_REF][START_REF] Ruff | Characteristics, distribution, origin, and significance of opaline silica observed by the Spirit rover in Gusev crater, Mars[END_REF][START_REF] Squyres | Detection of silica-rich deposits on Mars[END_REF]. Recent evidence suggests that the Home Plate opaline silica is most likely a siliceous hot spring deposit, or sinter, although the geochemistry of the original thermal fluids is currently unknown (Ruff, Campbell, Van Kranendonk, Rice & Farmer, 2019, in press). These deposits occur as nodular outcrops, some with digitate (finger-like) structures that resemble biomediated digitate sinter deposits among the hot spring and geyser discharge channels of El Tatio, Chile [START_REF] Ruff | Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile[END_REF].

Digitate structures similar to those at Home Plate and El Tatio are found at the air-water interface in the shallow discharge channels and around pool rims of many terrestrial hot springs, where spicular (needle-like) to knobby morphologic subtypes form under the influence of evaporation and wicking, and are characterized by silicified microorganisms within the fine-scale sinter laminae (cf. [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF][START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Handley | Silicifying biofilm exopolymers on a hot-spring microstromatolite: templating nanometer-thick laminae[END_REF]. In hot springs, alternating layers of mineralized microbial biofilms and silica build up over time to produce microstromatolites, including spicular, knobby and (pseudo)columnar geyserite, which are akin to layered structures prevalent in early Earth paleoenvironments (Campbell et al., 2015a;Djokic, Van Kranendonk, Campbell, Walter, & Ward, 2017;Guido, Campbell, Fouchet, & Westall, 2019;[START_REF] Handley | Character, analysis, and preservation of biogenicity in terrestrial siliceous stromatolites from geothermal settings[END_REF][START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF]Murphy et al., in press).

Rapid silicification of microbes by amorphous opal-A has a high potential to preserve microbial cells as biosignatures (e.g., [START_REF] Handley | Character, analysis, and preservation of biogenicity in terrestrial siliceous stromatolites from geothermal settings[END_REF][START_REF] Konhauser | Microbial-silica interactions in Icelandic hot spring sinter: Possible analogues for some Precambrian siliceous stromatolites[END_REF], although the more subtle biofilm horizons produced by some microbial communities and incorporated into sinter do not leave obvious macroscopic traces of cells. These require additional methods to illuminate their existence, such as electron microscopy or Raman spectroscopy (e.g., [START_REF] Guido | Life is everywhere in sinters: examples from Jurassic hot spring environments of Argentine Patagonia[END_REF][START_REF] Handley | Silicifying biofilm exopolymers on a hot-spring microstromatolite: templating nanometer-thick laminae[END_REF]. Without further diagenesis (i.e. the re-mineralization or dissolution of opal-A), the textures of fossilized microorganisms may be preserved within the microstromatolites, and thus serve as excellent records of past biological activities [START_REF] Cady | Fossilization processes in siliceous thermal springs: Trends in preservation along thermal gradients[END_REF][START_REF] Cady | Chapter 7 -Siliceous hot spring deposits: Why they remain key astrobiological targets[END_REF]Campbell et al., 2015a;[START_REF] Campbell | Tracing biosignature preservation of geothermally silicified microbial textures into the geological record[END_REF][START_REF] Farmer | Hydrothermal systems: Doorways to early biosphere evolution[END_REF][START_REF] Handley | Character, analysis, and preservation of biogenicity in terrestrial siliceous stromatolites from geothermal settings[END_REF].

Even with diagenesis and metamorphism, sinter may preserve evidence of past biological activity for up to billions of years (e.g., [START_REF] Djokic | Earliest signs of life on land preserved in ca. 3.5 Ga hot spring deposits[END_REF].

Previous studies showed that microbial cells and biofilms affect silica nucleation and the morphological development of sinter deposits. [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF] demonstrated that microbial colonies form the nuclei of spicular sinter that grows around the rim of Champagne Pool (Wai-o-Tapu, New Zealand), and that repeated surface colonization and biofilm silicification (particularly at spicule tips) results in the internal layering and development of spicular sinter structures.

Amorphous silica can rapidly fossilize filamentous colonies, forming minute micron-scale digitate sinter structures within 72 hours [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF]. Moreover, exopolymeric substances (EPS), which biofilm-forming microbes produce for survival (e.g., for nutrient and enzyme facilitation, toxin exclusion and substrate attachment), can also be silicified (e.g. [START_REF] Krumbein | Fossil and recent biofilms: A natural history of life on Earth[END_REF]. Eventually, layers of mineralized microbial cells and EPS build up to form microstromatolites, as shown by experiments generating a spectrum of unsilicified to heavily silicified hot spring biofilms, which identified homogenous solid silica laminae as silicified EPS [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Handley | Silicifying biofilm exopolymers on a hot-spring microstromatolite: templating nanometer-thick laminae[END_REF]. Evaporative wicking, combined with capillary action, has been shown to be the main driver of silica nucleation upon microbial materials in subaerial sinters [START_REF] Cady | Fossilization processes in siliceous thermal springs: Trends in preservation along thermal gradients[END_REF][START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF][START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF].

Although the presence of microbial cells is important for the overall build-up of digitate sinter [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Handley | Silicifying biofilm exopolymers on a hot-spring microstromatolite: templating nanometer-thick laminae[END_REF], it is currently unknown if the taxonomic groupings of the microbes or their cellular shapes contribute to the morphologic variations of digitate sinter. Moreover, although digitate sinters have been shown to be coated in, and incorporate, abundant microorganisms (e.g. [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Jones | Vertical zonation of biota in microstromatolites associated with hot springs, North Island, New Zealand[END_REF][START_REF] Jones | Petrography and genesis of spicular and columnar geyserite from the Whakarewarewa and Orakeikorako geothermal areas, North Island, New Zealand[END_REF]2006;[START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF], a systematic characterization of these microbial communities has not previously been conducted. Here we explored microbial communities associated with modern digitate sinters that were actively forming in different geothermal fields within the Taupo Volcanic Zone (TVZ), New Zealand. The aims of this study were to examine (1) the microbial communities (bacterial, archaeal and eukaryotic) associated with TVZ digitate sinters growing in a wide range of physicochemical conditions; (2) the factors that control these microbial communities; and (3) the microbiological or abiotic factors associated with distinct digitate sinter morphologic subtypes. Our results show that microbial community composition is strongly controlled by water chemistry, degree of subaerial exposure, pH, and temperature. Diverse microorganisms, and cell shapes and sizes, contribute to the overall build-up of digitate sinter macrostructures. However, results suggest that hydrodynamics plays a more important role than microbiology in creating digitate sinter subtypes.

Throughout this study, the usage of the term 'digitate' (finger-like) to describe sinter morphology was adopted from [START_REF] Ruff | Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile[END_REF]. The term appears to have been introduced by [START_REF] Braunstein | Relationship between spring and geyser activity and the deposition and morphology of high temperature (> 73 degrees C) siliceous sinter, Yellowstone National[END_REF], who used it to describe high-temperature, geyseritic sinter growing horizontally poolward from the rims of non-surging hot pools at Yellowstone National Park (U.S.A.), and which ranges in morphology from finger-like to lily pad-shaped. [START_REF] Campbell | An unusual modern silica-carbonate sinter from Pavlova spring, Ngatamariki, New Zealand[END_REF] described somewhat similar features at Pavlova Spring (TVZ, New Zealand), although these contained minor calcite and were described as meringue-like in form. Here, we describe all morphotypes of vertical sinter protrusions from the substrate as digitate sinters, sensu [START_REF] Ruff | Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile[END_REF]. The height-to-width aspect ratio of these features in the TVZ was used to define subtypes of digitate sinter morphology, where: (1) 'spicular' indicates the height of each protrusion was greater than its width, (2) 'bulgy' indicates protrusions had similar heights and widths, and (3) 'knobby' signifies the width of each protrusion was greater than its height (Table 1). In addition, the spicular subtype comprises fine needle-like protrusions, as well as wide protrusions that are referred to as (pseudo)columnar. Stromatolites, including siliceous hot spring forms, "are laminated organosedimentary structures built by microscopic algae and bacteria" [START_REF] Walter | Siliceous algal and bacterial stromatolites in hot spring and geyser effluents of Yellowstone National Park[END_REF]. We use the terms stromatolite or microstromatolite when referring to the TVZ digitate sinters, as previously used by [START_REF] Jones | Vertical zonation of biota in microstromatolites associated with hot springs, North Island, New Zealand[END_REF][START_REF] Farmer | Hydrothermal systems: Doorways to early biosphere evolution[END_REF] to describe similar structures on the basis that they are internally laminated at the micron-scale with layers that are alternatively dense and mineral-rich or porous, and rich in fossilized microorganisms. This definition also includes silicification of biomolecules (e.g. lipids), which may not be visually evident but which can be revealed through various analytical methods such as Raman mapping and lipid biomarker analysis (e.g. [START_REF] Kaur | Preservation of microbial lipids in geothermal sinters[END_REF][START_REF] Kaur | Microbial membrane lipids in active and inactive sinters from Champagne Pool, New Zealand: elucidating past geothermal chemistry and microbiology[END_REF].

Materials and methods

Site description, sample collection, and physicochemical measurements of water

Different subtypes of digitate sinter from 11 hydrothermal pools and discharge channels in active geothermal fields of the TVZ, New Zealand, were sampled in April 2018. Each sampling site consisted of five spatial replicates (0.5 to 1 m apart). Sampled geothermal features were Orange Spring, Te Kopia, Parariki Stream, Rotokawa, Tikitere and Whangapaoa Spring (Fig. 1 and Table 1).

Actively growing, digitate siliceous sinter samples, spicular (fine, wide, bulgy) or knobby in morphology, were collected aseptically, kept in sterile 50 mL polypropylene tubes (Greiner Bioone, Germany), transported on dry ice, and stored at -80°C until further processing. Water temperatures were recorded in situ using a COMARK Evolution IV9001 temperature probe (Norfolk, UK), and pH values were recorded using the portable sension™156 Multiparameter Meter (Hach Company, USA). Sinters were photographed, their dimensions measured and their macro-morphologies characterized based on criteria from [START_REF] Walter | Microbiology and morphogenesis of columnar stromatolites (Conophyton, Vacerrilla) from hot springs in Yellowstone National Park[END_REF] and [START_REF] Walter | Proterozoic stromatolites[END_REF].

Scanning electron microscopy

In preparation for scanning electron microscopy (SEM) analysis, frozen digitate samples were thawed, fractured into 0.5-1.0 cm pieces, including cross-sectioning, and fixed overnight in 2.5% glutaraldehyde. Fragments were dehydrated by soaking in ethanol solutions of increasing concentration (50%, 70%, 80%, 90% and 100%, with each step repeated twice). Samples were then placed in a Polaron ED3000 Critical Point Drier (Polaron, Watford, UK) to replace ethanol with liquid CO2. Critical-point dried samples were stored in a sealed vacuum container filled with silica gel until studied via SEM. Before imaging, samples were mounted onto SEM stubs and coated with platinum using a Quorum Q150RS rotary pumped sputter coater (Quorum Technologies Ltd, UK). A FEI QUNATA 200F environmental SEM was used for imaging. Operating conditions were 10-20 keV, with a pressure of ~0.5 torr, and a working distance of ~10 mm.

Microbial community analysis

Total DNA was extracted from 0.25 g of each homogenized digitate sample (only the subaerial, protruding part) using the DNeasy PowerSoil® Kit (Qiagen, USA) following the manufacturer's instructions. The amount and quality of extracted gDNA were checked using a NanoPhotometer (Implen, Germany) and Qubit 3.0 fluorometric quantitation (Invitrogen, USA). Bacterial and archaeal 16S rRNA genes were amplified using the 515'F (GTGBCAGCMGCCGCGGTAA) forward primer [START_REF] Hugerth | DegePrime, a program for degenerate primer design for broad-taxonomicrange PCR in microbial ecology studies[END_REF] and 926'R (CCGYCAATTYMTTTRAGTTT) reverse primer [START_REF] Walters | Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys[END_REF]. Eukaryotic 18S rRNA genes were amplified using primers CDMF/CDMR (GTCAGAGGTGAAATTCTTGGATTTA and AAGGGCAGGGACGTAATCAACG) [START_REF] Harvey | Using PCR Amplification and Genetic Sequence Analysis of 18S rRNA Genes to Survey the Microbial Diversity and Distribution of Eukaryotic Microbes Inhabiting Two Thermo-acidic Streams in Yellowstone National Park, Wyoming[END_REF], and Euk1391f/EukBr (GTACACACCGCCCGTC and TGATCCTTCTGCAGGTTCACCTAC) (Amaral-Zettler, [START_REF] Amaral-Zettler | A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA Genes[END_REF]. All primer pairs were synthesized with Illumina adapters (Document # 1000000002694; Illumina, USA). Polymerase chain reaction (PCR) was performed in 25 µL volumes containing 12.5 µL of MyTaq HS Red Mix, 2x (Bioline, UK), 0.5 µL of each primer (20 µM), 5 µL of template DNA and 6.5 µL of nuclease-free water. The PCR conditions for 16S rRNA gene amplification were 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 55.5°C for 30 seconds and 72°C for 30 seconds. The PCR conditions for 18S rRNA amplification using the CDMF/CDMR primers were 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 60°C for 1 minute and 72°C for 1 minute. PCR conditions for the Euk1391f/EukBr primers were 94°C for 3 minutes, 35 cycles of 94°C for 45 seconds, 57°C for 1 minute and 72°C for 90 seconds. All cycling conditions were followed by a final elongation step of 72°C for 10 minutes. Gel electrophoresis (1% w/v, 1X TBE agarose gel, running at 80 V for 45 minutes) was undertaken to check the size and quality of PCR products. Agencourt AMPure XP magnetic beads (Beckman Coulter, USA) were used to purify PCR products and the Qubit fluorometer was used to quantify DNA concentrations.

Barcoded libraries were prepared according to Illumina's 16S Metagenomic Sequencing Library Preparation manual. 5 µL of each indexed amplicon was pooled and cleaned-up using Agencourt AMPure XP magnetic beads (Beckman Coulter, USA) using 1:1 ratio. Quality control of the library pool was checked using an Agilent BioAnalyzer (Integrated Sciences, Sydney, Australia) with a High Sensitivity DNA chip, and the quantification was checked using Qubit fluorometer. The library pool was diluted to 4 nM using the average size of the library in bp before being loaded onto the MiSeq sequencer. Multiplexed 2x250 bp paired-end Illumina sequencing was undertaken according to Illumina's MiSeq System Denature and Dilute Libraries Guide (Document #15039740 v06; Illumina, USA) and Illumina's MiSeq System Guide (Document #15027617 v04 Material #20024228; Illumina, USA). To improve diversity, libraries were loaded with 10% PhiX, at 8 pM using MiSeq Reagent Kit v2. Sequencing was undertaken at the Auckland Genomics, The University of Auckland, Auckland, New Zealand. QIIME2 (version 2018(version .8, Bolyen et al., 2018) ) was used to quality trim and analyze demultiplexed sequencing reads, by joining [START_REF] Rognes | VSEARCH: A versatile open source tool for metagenomics[END_REF][START_REF] Vázquez-Baeza | EMPeror: A tool for visualizing high-throughput microbial community data[END_REF], quality filtering (Q score cutoff of 25) (Bokulich et al., 2013), denoising [START_REF] Amir | Deblur rapidly resolves single-nucleotide community sequence patterns[END_REF], chimera filtering [START_REF] Rognes | VSEARCH: A versatile open source tool for metagenomics[END_REF][START_REF] Vázquez-Baeza | EMPeror: A tool for visualizing high-throughput microbial community data[END_REF], and generation of an amplicon sequence variants table [START_REF] Bokulich | Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2's q2-feature-classifier plugin[END_REF]. 16S and 18S rRNA gene amplicon reads have been deposited in the NCBI Sequence Read Archive database under BioProject PRJNA543937. Taxonomy assignment was conducted using the SILVA database version 132 [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF][START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF][START_REF] Yilmaz | The SILVA and "all-species Living Tree Project (LTP)" taxonomic frameworks[END_REF]. Relative abundance, alpha diversity and beta diversity were determined using R (version 3.4.4, R Core Team, 2017) with RStudio software (version 3.4.1, RStudio Team, 2015), and R packages ggplot2 (version 3.0.0, [START_REF] Wickham | ggplot2: elegant graphics for data analysis[END_REF]vegan (version 2.5.1, Oksanen et al., 2018).

Quantitative Droplet Digital PCR (ddPCR) was used to determine the absolute number of prokaryotes and eukaryotes in each sample. All ddPCR reactions were conducted in 22 µL volumes containing 11 µL of 2x QX200™ ddPCR™ EvaGreen® Supermix (BIO-RAD, USA), 0.5 µL of each primer (without Illumina adapters), 8 µL of nuclease-free water and 2 µL of DNA template. The BIO-RAD QX200 droplet generator was used to generate 40 µL of droplet PCR mixtures. ddPCR conditions were similar to those for PCR but with additional signal stabilization steps of 4°C for 5 minutes and 90 °C for 5 minutes after the final extension, and amplification was undertaken using an Applied Biosystems® Veriti® 96-Well Thermal Cycler. Amplified ddPCR products were quantified using a BIO-RAD QX200 droplet reader and analyzed using QuantaSoft™ Analysis Pro software (version 1.0) in order to obtain the copy number of each gene.

Results

Site description, physicochemical properties of spring waters, and digitate sinter characterization

Descriptions of sites including pH and temperature of the thermal waters, and digitate sinter morphologic subtypes are given in Table 1. Based on published literature, spring water chemistry was characterized as acid-sulfate (sites OR, TKP1 and TKP2), acid-sulfate-chloride (sites PA, RTB, RTG, RTSH and TIK1), acid-sulfate-bicarbonate (sites TIK2 and TIK3), and alkali-chloride (site WH) (Jones & Renault, 2007;[START_REF] Power | Microbial biogeography of 925 geothermal springs in New Zealand[END_REF][START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF]unpublished data). A scatter plot showing physicochemical properties for each sample is shown in figure 2. A temperature of 34.5°C was the lowest average spring temperature and was measured at an acid-sulfatebicarbonate spring at Tikitere (pool and channel TIK3). The highest average spring temperature (86.9°C) was recorded at TKP2, an acid-sulfate pool at Te Kopia. The most acidic site was PA at Parariki Stream (pH 1.0), and the most alkaline pool was WH at Whangapaoa (pH 8.5). At the study sites, siliceous digitate sinter was found forming subaerially at the air-water interface, either in spring discharge channels above shallow sheet flow or around the innermost edges of pools (e.g. Fig. 3; Supplementary figures 1-7). Sinter branching morphologies were mainly multifurcate (i.e. divided into multiple branches), while site TIK3 sinters were double-multifurcate (i.e. additional branching from prior divisions). Digitate sinter protrusions clustered into lumps from a few millimetres to centimetres in width (Fig. 3b,3d,3f and 3h). Each individual fine spicular digitate sinter protrusion was ~0.4-1.1 mm wide and ~0.8-1.4 mm high, while wide spicular protrusions were ~1.5 mm wide and 2.5-4.0 mm high, and bulgy spicules were ~1.0-1.1 mm in both height and width. Digitate samples from Whangapaoa Spring were the least spicular in shape, forming knobby protrusions (~8.2 mm wide and 3.7 mm high), where the height of each protrusion was much less than its width, and each individual protrusion was much larger than that of spicular digitate sinters. Overall, digitate sinter protrusions were light brown, beige, grey or white in colour, and the submerged bases of some sinters were coated in colourful microbial mats at both acidic (dark green) and alkaline sites (dark green or red).

Features of digitate sinter among hot spring sites

Digitate sinter morphological variations

Each spring appeared to have digitate sinters with distinct morphologies (Fig. 4a, 4d, 4g and4j).

Digitate sinter morphologic subtypes were consistent within a spring, regardless of whether sinters were collected from pool rim or channel locations. A relationship between morphologic subtype and hydrodynamic activity was apparent (Table 1). Fine, spicular digitate sinters were observed to grow where the water flow is calm and quiet, normally in the very shallow (mm's to a few cm's depth) outflow areas of pools, which in this study were all acidic (pH <7). Wide or bulgy spicular digitate sinters were observed to form where ripples or splashes occasionally bathed the sinter at the rim of acidic pools. It is worth noting that although fine and wide spicular digitate sinters grow in springs with similar physicochemistries, they were never observed together in the same spring. Knobby digitate sinter was present only in the gentle outflow of a single high pH, alkali-chloride spring in this study. Digitate sinter subtypes defined here also varied slightly in shape and colour, for example, those observed at the pools and channels at site TIK2 compared to TIK3, which are situated only about a metre apart from one another. Digitate sinter from TIK2 hosted spicules in a multifurcate arrangement and were beige in colouration, whereas digitate sinter from TIK3 was double-multifurcate and had grey colour.

Microbial laminations observed in digitate sinter

Silica laminae were evident in digitate sinter interiors from each spring, with combinations of porous and solid laminae generating distinct microtextures within and across the samples (Fig. 4b, 4e, 4h and 4k). Mineralized microbial cells were clearly present within sinter cross-sections (Fig. 4c, 4f, 4i, 4l; Supplementary figures 8-9), and have been suggested shown elsewhere to contribute to the texture and build-up of laminae [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Handley | Silicifying biofilm exopolymers on a hot-spring microstromatolite: templating nanometer-thick laminae[END_REF].

Newer layers of biofilm forming on the outer tips of digitate sinters appeared only partially silicified. Overall, broken cross-sections of digitate sinter from different springs showed varied levels of porosity. However, no clear correlation was apparent between spring chemistry and differences in microbial laminations.

Dominant microbial cell shapes and sizes

Under low magnification in SEM, silicified biofilms were observed to cover sinter surfaces. Higher magnification SEM imaging revealed a range of silicified microorganisms (Fig. 4c, 4f, 4i and 4l), in particular, prokaryotes, acidophilic algae, and also other eukaryotes, including insect eggs and larvae. Several microbial cell shapes were observed. At acid-sulfate springs, such as Orange Spring, the dominant cell shapes observed were rod-shaped prokaryotes and larger coccoidal algal (Cyanidiophyceae) cells (Fig. 4c). Coccoidal algal cells also were found to be dominant at acid-sulfate-chloride springs, such as Parariki Stream (Fig. 4f) (see also [START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF]. At higher pH, acid-sulfate-bicarbonate springs, most cells were either rod-shaped or filamentous prokaryotes (Fig. 4i and supplementary figure 8). Finally, filamentous prokaryotes and eukaryotes were both abundant at the Whangapaoa alkali-chloride spring (Fig. 4l). Although each spring chemistry type seemed to favour particular collections of microbial cells, we assessed each cell shape from similarly magnified SEM micrographs and found that there was no apparent relation between the dominant cell shapes and the pattern of microbial lamination or digitate sinter morphologic subtype.

Community analyses of prokaryotes and eukaryotes in digitate sinter

Community differences across environmental gradients

Amplicon sequences of phylogenetic marker genes were analyzed in order to compare microbial communities across sinters from different springs. Water chemistry (in terms of main ions present), along with pH and temperature, were observed to be strong predictors of community composition (Fig. 5a-c). Both pH and temperature were significant variables influencing prokaryotic community composition and structure (Fig. 5a). In particular, a high level of dissimilarity was evident between alkaliphilic (alkali-chloride) and acidophilic (acid-sulfate type) communities. Among the acidic spring types, acid-sulfate and acid-sulfate-chloride springs tended to have more shared prokaryotic communities than either spring type had with acid-sulfatebicarbonate springs. Similar trends in beta diversity were observed for prokaryotes and eukaryotes. However, the level of dissimilarity among eukaryotic communities from different spring geochemistries was much greater than those among prokaryotic communities (Figs 5b-c versus 5a).

Community composition in digitate sinter

16S rRNA gene reads were classified into 26 phyla, which were dominated by Euryarchaeota (33.31% average relative abundance), Proteobacteria (mostly Gammaproteobacteria and Alphaproteobacteria) (20.92%), Crenarchaeota (19.94%), Cyanobacteria (5.12%) and Bacteroidetes (4.28%) (Fig. 6a). At high temperature (>70°C) and highly acidic (pH<3) springs, two archaeal phyla, Crenarchaeota and Euryarchaeota, dominated more than three-quarters of each community (53.01% and 25.29% respectively), followed by Proteobacteria (14.29%). At moderate temperature (~40-70°C) springs with similarly low pH values, the relative abundance of Euryarchaeota was almost three times higher (72.83%), and very few Crenarchaeota were detected. However, the relative abundance of acidophilic Proteobacteria was comparable across most of the temperature gradient at low pH.

At most circumneutral and alkaline pools, almost no archaea were detected, regardless of temperature. Proteobacteria were consistently abundant at these sites, and were the most abundant phylum (52.83%) at acid-sulfate-bicarbonate springs. However, at moderate to moderately high temperature (~40-70°C) springs with neutral pH, but with acid-sulfate-chloride chemistry, Euryarchaeota was the most abundant taxon (45.19%) -similar to springs with low pH and moderate temperatures. Taxa associated with digitate sinter from the moderate to moderately high temperature (~40-70°C), alkali-chloride spring (pH>8) were mostly distinct from those found in sinters associated with circumneutral pHs. Of these, Cyanobacteria had the highest relative abundance (34.76%), followed by Bacteroidetes (16.88%) and Chloroflexi (14.18%).

Across 55 digitate sinter samples, Acidithiobacillus (Gammaproteobacteria) and A-plasma (Euryarchaeota) were the most widespread taxa, with 83.63% and 70.91% prevalence, respectively. Both of these were found in almost every acidic and circumneutral pH spring, but neither was detected in the alkali-chloride spring.

To determine the similarity of microbial communities associated with the same pool chemistries, but different sinter macro-morphology types, a total of nine other samples were collected at Parariki Stream from three different non-digitate sinter types (Supplementary figures 10-12). Two of these sinter types, cup-shaped (PAC) and sheet-shaped (PASH), were subaerial at the time of collection, although previous study indicates that sheet sinter develops under alternating wet (thin sheet flow of thermal waters) and dry conditions [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF]. Cup-shaped sinter grows on sandy substrates positioned at higher elevations above the floodplain compared to the other sinter types at Parariki Stream, while digitate sinter largely grows on pumice clasts [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF]. Both cup and digitate sinters accumulate near the air-water interface, with the cup-shaped sinter positioned at higher elevations (few cm's) above water-saturated sandy substrates. In contrast, the brown, vent-related sinter with irregular ridges and cavities (PAB) was sampled from a submerged zone in the vent area, under the highest temperature conditions under which sinter forms at Parariki Stream. These varied sinter fabrics are described in detail by [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF] and [START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF]. The three subaerial or occasionally subaerial sinter types from Parariki Stream (cup, sheet and digitate sinters) shared similar microbial communities, which were also shared with digitate sinters from other highly acidic springs (Fig. 6a and6b). In contrast, the microbial composition of vent-related sinter (PAB) from the Parariki Stream hot springs area was distinct, reflecting the unique submerged and high-temperature habitat. This vent deposit was coated in a thin brown biofilm and was colonized solely by Crenarchaeota, particularly Sulfolobales and Aigarchaeales. This result from Parariki Stream shows that different sinter morphologies (cup, sheet and digitate) can provide equivalent microbial habitats, and may therefore develop from essentially the same types of microbial cells, albeit under slightly different physical environmental conditions. The contribution of similar or identical assemblages of microbial communities to the formation of distinct sinter macro-morphologies is consistent with studies showing that microbial cells are passive substrates upon which silica precipitates [START_REF] Benning | Biosilicification: the role of cyanobacteria in silica sinter deposition[END_REF][START_REF] Mountain | Experimental studies on New Zealand hot spring sinters: rates of growth and textural development[END_REF]Phoenix, Adam & Konhauser, 2000;[START_REF] Yee | The effect of cyanobacteria on silica precipitation at neutral pH: implications for bacterial silicification in geothermal hot springs[END_REF].

Over the 11 sampling localities, 18S rRNA gene reads, amplified by primers CDMF/CDMR, were classified into five phyla/groups, which were dominated by Archaeplastida (75.66%), the SAR supergroup (14.39%) and Opisthokonta (6.75%) (Fig. 7a). Non-amplifiable samples were excluded.

All highly acidic sinters (pH <3) of moderate to high temperatures (>40°C) were mostly populated by Archaeplastida (red algae) (>90%). However, when pH increased, Opisthokonta comprised half of the community (48.84%), along with Archaeplastida (47.25%), demonstrating that members from Archaeplastida detected in this study (Cyanidiophyceae) were hyperacidophilic (Supplementary figure 1415). At low temperature with circumneutral pH, the abundance of acidophilic Archaeplastida decreased to less than a quarter of the community, and SAR supergroups dominated instead, followed by Amoebozoa and Opisthokonta. Amplicons from knobby digitate sinter in alkali-chloride pool WH were only amplified using the Euk1391f/EukBr primers, due to the presence of a distinct group of alkaliphilic microorganisms (Fig. 7b and Supplementary figure 15). The CDMF/CDMR primers were designed specifically for acidophilic microbial communities and failed to amplify DNA from sinter samples at site WH. For comparison to WH, samples from acidic springs RTB, RTG, RTSH, TIK1, TIK2 and TIK3 were also amplified with the Euk1391f/EukBr primer set. However, extracted gDNA from OR, PA, TKP1 and TKP2 sites failed to be amplified by Euk1391f/EukBr, along with spatial replicates from some other sites. Results

showed that almost 90% of sequences derived from WH were from Opisthokonta, specifically Ecdysozoa and Dorylamia.

Diversity and quantification of prokaryotes and eukaryotes in digitate sinter

Shannon indices revealed similar overall trends in prokaryotic and eukaryotic diversity related to temperature and pH (Fig. 8a-8d). A statistically significant negative correlation was found between temperature and prokaryotic diversity (R 2 =-0.43, p=0.0012). This remains true when considering only bacterial diversity (R 2 =-0.57, p=0.0000069). On the other hand, pH exhibited a strong positive correlation with overall prokaryotic diversity (R 2 =0.5, p=0.00011), and bacteria alone (R 2 =0.46, p=0.0004). Archaeal communities displayed the opposite trends in both cases, and were positively correlated with temperature (R 2 =0.63, p=0.00000032), but negatively correlated with pH (R 2 =-0.52, p=0.000046). Eukaryotic community trends were similar to bacteria when considering acidophilic primers (CDMF/CDMR), while the general eukaryotic primers (Euk1391f/EukBr) revealed no trend with pH.

Quantification by ddPCR revealed that there were more prokaryotes than eukaryotes in most digitate sinters from highly acidic waters with temperatures ranging from moderate to high (Fig. 9). The exception was Orange Spring, where eukaryotes, identified as Cyanidiophyceae by SEM and sequencing, were more abundant than prokaryotes. Digitate sinter from circumneutral pH (TIK2, TIK3) and alkaline springs (WH) -all with moderate temperatures -had a higher number of prokaryotes than at other sites. At two of these sites (TIK2 and WH) the number of eukaryotes was also elevated, and equivalent to prokaryotes. As for other microbiological attributes, no correlation was evident between microbial abundance or diversity and digitate sinter morphology.

Discussion

Morphological variation of digitate sinter and its relationship with hydrodynamics and physicochemistry

New Zealand's hot springs have been shown to have broad ranges of pH, temperature, and water geochemistry [START_REF] Power | Microbial biogeography of 925 geothermal springs in New Zealand[END_REF]. Our results show that digitate sinters, in particular, also develop under a broad range of physicochemical conditions of the springs, although they were found to consistently form at the air-water interface in shallow discharge channels or around spring-pool rims.

Digitate sinter in this study comprised two main morphological end-members -knobby and spicular. Each end-member formed under distinct physicochemical and hydrodynamic conditions, consistent with observations elsewhere. Knobby sinter was found in gently flowing alkali-chloride waters at Whangapaoa (Table 1), and has been shown to form under similar conditions at El Tatio, Chile [START_REF] Ruff | Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile[END_REF] and Pong Duet Pa Pae (Thailand) (Supplementary figure 18). In comparison, fine spicular sinter grew at several locations in this study with gently flowing acidic and circumneutral pH waters, although major ions present varied (Table 1). Fine spicules have also been observed at other comparable locations in both the TVZ, such as Champagne Pool and Kerosene Creek (Wai-O-Tapu, New Zealand), Orakei Korako (New Zealand), Whakarewarewa (New Zealand), and elsewhere, such as Whirligig geyser outflow (YNP,USA) (Table 1; Supplementary figures 16-17) [START_REF] Campbell | Tracing biosignature preservation of geothermally silicified microbial textures into the geological record[END_REF][START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF][START_REF] Jones | Petrography and genesis of spicular and columnar geyserite from the Whakarewarewa and Orakeikorako geothermal areas, North Island, New Zealand[END_REF][START_REF] Jones | Vertical zonation of biota in microstromatolites associated with hot springs, North Island, New Zealand[END_REF][START_REF] Lynne | Mapping vent to distal-apron hot spring paleo-flow pathways using siliceous sinter architecture[END_REF][START_REF] Walter | Microbiology and morphogenesis of columnar stromatolites (Conophyton, Vacerrilla) from hot springs in Yellowstone National Park[END_REF]. Interestingly, fine spicular digitate sinter has also been found at the continuously splashing and boiling zones of very ebullient alkali springs or geysers, for example, Diamond geyser (Orakei Korako), Steep cone spring (YNP) [START_REF] Braunstein | Relationship between spring and geyser activity and the deposition and morphology of high temperature (> 73 degrees C) siliceous sinter, Yellowstone National[END_REF][START_REF] Lynne | Mapping vent to distal-apron hot spring paleo-flow pathways using siliceous sinter architecture[END_REF], and Pong Duet Pa Pae (Thailand) (Supplementary figure 19). While fine spicular sinters at ebullient alkali sites seem to share the same morphologic subtype with those in calm acidic waters, those at alkali sites appear generally much smaller in size (Supplementary figure 19).

Besides the fine spicular sinter subtypes identified in this study, wide or bulgy digitate sinter subtypes were present in acidic to circumneutral pH TVZ locations as well. However, the wide or bulgy subtypes were subjected to occasional, moderate splashing rather than quiet waters (Table 1). The bulgy subtype, which was found only at TKP2, may represent a transition between wide spicular digitate sinter to cup-shaped sinter. Digitate sinter in the TVZ can also grow alongside other kinds of sinter microfacies, for example, sheet-and cup-shaped sinter, as discussed above [START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF]. Accordingly, the wide spicular subtype was also present at TKP2, but was not sampled owing to its physically intercalated growth with cup-shaped sinter.

Taken together, our field observations and subsequent laboratory analyses suggest that hydrodynamics influence digitate sinter morphologies, producing a range of subtypes. Spring hydrodynamics have previously been shown to play a major role in shaping diverse sinters [START_REF] Braunstein | Relationship between spring and geyser activity and the deposition and morphology of high temperature (> 73 degrees C) siliceous sinter, Yellowstone National[END_REF], and [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF] demonstrated that micro-environmental physical effects (e.g., wave activity, wind direction, and steam-condensation) influence the morphology and growth rate of biomediated digitate sinter in Champagne Pool. As gently flowing hot spring waters were found to be associated with both spicular (pH <7) and knobby (pH >7) sinter subtypes under distinct pH ranges, results suggest physicochemistry may also influence digitate subtypes to a limited extent. However, with only three examples of knobby sinter identified (Table 1) further work is needed to confirm this association.

Relationship between digitate sinter morphology and microbiology

When silica precipitates in hot springs as amorphous opal-A, it can preserve microorganisms and their products (e.g., EPS) through silicification, a process whereby their cell walls and cytoplasmic materials are permineralized [START_REF] Cady | Fossilization processes in siliceous thermal springs: Trends in preservation along thermal gradients[END_REF]. Under fluctuating environmental conditions, especially wetting and drying of the sinter apron, the cumulative accretion of silicified laminae eventually forms microstromatolites, which grow, at least in part, by evaporative wicking at the air-water interface [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF]2008). Although microorganisms physically contributevia cell growth and accumulation -to the build-up of layers that make up digitate sinters [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF]2008), we found no evidence suggesting that microbial taxa composition influenced the morphologic subtype of digitate sinter, as similar subtypes were produced where microbial community compositions were distinct. For example, OR and PA pools still had the same subtype of fine, spicular digitate sinter, although OR pool had a substantially greater proportion of Proteobacteria (Fig. 6a). We also found no relation between the dominant cell shapes of microorganisms colonizing digitate sinters and the pattern of internal lamination. A lack of correlation between digitate sinter morphologic subtypes and cell shape may be expected, given that the role of microbial cells in the build-up of microstromatolites is thought to be a passive process, whereby they facilitate silica deposition by providing functional groups and metabolites that are ideal for silica nucleation [START_REF] Konhauser | Introduction to Geomicrobiology[END_REF][START_REF] Lalonde | The experimental silicification of Aquificales and their role in hot spring sinter formation[END_REF]Phoenix, Konhauser & Ferris, 2003). This does not, however, imply that the presence of microbial communities is not an important contributing factor in the development and build-up of microstromatolites in digitate sinter as they constitute a considerable proportion of the physical bulk within any given deposit. Moreover, previous work identifying focused microbial colonization and silicification at spicular sinter tips, provides strong evidence that the characteristic finger-like shape of these sinters is biomediated [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF]. Further laboratory study outside the scope of this current contribution must be undertaken to determine whether digitate structures can form by evaporative wicking, splash or spray in the absence of microorganisms.

Prokaryotic communities associated with microstromatolites in TVZ hot springs

While this study included siliceous (predominately digitate) hot spring microstromatolites from a wide range of physicochemistries, most studies of freshwater hot spring stromatolites elsewhere outside New Zealand have focused on those that grow in circumneutral or alkali-chloride springs [START_REF] Farías | The discovery of stromatolites developing at 3570 m above sea level in a high-altitude volcanic lake Socompa, Argentinean Andes[END_REF][START_REF] Konhauser | Microbial-silica interactions in Icelandic hot spring sinter: Possible analogues for some Precambrian siliceous stromatolites[END_REF][START_REF] Reyes | Surface orientation affects the direction of cone growth by Leptolyngbya sp strain C1, a likely architect of coniform structures Octopus Spring (Yellowstone National Park)[END_REF][START_REF] Schuler | Hot spring microbial community composition, morphology, and carbon fixation: Implications for interpreting the ancient rock record[END_REF][START_REF] Toneatti | Stratified bacterial diversity along physico-chemical gradients in high-altitude modern stromatolites[END_REF].

Nevertheless, stromatolite communities from several geographically diverse circumneutral and alkaline hot spring locations where found to share taxa with digitate sinters from physicochemically similar springs in the TVZ (circumneutral, acid-sulfate-bicarbonate pools TIK2 and TIK3, and the alkali-chloride pool WH). This includes the Great Obsidian Pool Area (GOPA) of YNP (pH=6.28, temperature=48.4°C), Octopus Spring of YNP (pH=8.3-8.8, temperature=22-59°C), an Argentinean volcanic lake (pH=6.5, temperature=26°C), and an Icelandic hot spring (pH=8.0-8.8, temperature=30-40°C). Shared taxa notably include Deinococci, Acidobacteria, Sphingobacteria, Cyanobacteria, Anaeroalineae, Thermoprotei and Thermoplasmata (Supplementary figure 13) [START_REF] Farías | The discovery of stromatolites developing at 3570 m above sea level in a high-altitude volcanic lake Socompa, Argentinean Andes[END_REF][START_REF] Konhauser | Microbial-silica interactions in Icelandic hot spring sinter: Possible analogues for some Precambrian siliceous stromatolites[END_REF][START_REF] Reyes | Surface orientation affects the direction of cone growth by Leptolyngbya sp strain C1, a likely architect of coniform structures Octopus Spring (Yellowstone National Park)[END_REF][START_REF] Schuler | Hot spring microbial community composition, morphology, and carbon fixation: Implications for interpreting the ancient rock record[END_REF][START_REF] Toneatti | Stratified bacterial diversity along physico-chemical gradients in high-altitude modern stromatolites[END_REF]. Of these, cyanobacteria were the dominant feature of most TVZ alkali-chloride Whangapaoa sinters. The second most abundant cyanobacterial genus at Whangapaoa, Leptolyngbya, was previously identified in partially lithified coniform structures from the alkali Octopus Spring at YNP [START_REF] Reyes | Surface orientation affects the direction of cone growth by Leptolyngbya sp strain C1, a likely architect of coniform structures Octopus Spring (Yellowstone National Park)[END_REF]. Moreover, although a relatively minor part of the Whangapaoa microstromatolite community, the cyanobacterial genus Calothrix has also been cultivated previously from a sulfate-chloride hot spring stromatolite in Krysuvik, Iceland [START_REF] Konhauser | Microbial-silica interactions in Icelandic hot spring sinter: Possible analogues for some Precambrian siliceous stromatolites[END_REF]. Although the dominant cyanobacterial genus at Whangapaoa, Synechococcus, was not documented in these other studies of alkali-chloride stromatolites, it is found in many non-stromatolitic microbial mats from alkali-chloride hot springs [START_REF] Becraft | Fine-scale distribution patterns of Synechococcus ecological diversity in microbial mats of mushroom spring, Yellowstone National Park[END_REF][START_REF] Cady | Fossilization processes in siliceous thermal springs: Trends in preservation along thermal gradients[END_REF][START_REF] Klatt | Community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential[END_REF][START_REF] Ward | Cyanobacterial ecotypes in the microbial mat community of Mushroom Spring (Yellowstone National Park, Wyoming) as species-like units linking microbial community composition, structure and function[END_REF].

In contrast, we found little evidence of shared communities when comparing non-stromatolitic, subaerial, circumneutral sinter mounds to circumneutral TVZ microstromatolite communities.

Sinter mound communities from El Tatio, Chile (pH=6.41-7.10, temperature=84°C) have a high abundance of Firmicutes and Alphaproteobacteria [START_REF] Sanchez-Garcia | Microbial biomarker transition in high-altitude sinter mounds From El Tatio (Chile) through different stages of hydrothermal activity[END_REF], while the studied TVZ circumneutral microstromatolite communities were dominated by either Thermoplasmata or Gammaproteobacteria (Supplementary figure 13). This difference may be due to the more pronounced wetting-drying cycles affecting the sinter mounds in Chile, perhaps resulting in a community more tolerant of longer periods of drying and UV exposure.

To date, siliceous stromatolites forming in highly acidic hot springs (pH <3) have been documented mainly in New Zealand (e.g. [START_REF] Jones | Stromatolites forming in acidic hot-spring waters, North Island, New Zealand[END_REF], and very few studies have determined their microbial composition [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF][START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF]. Our study explicitly reveals novel data on microbial communities associated with an array of siliceous stromatolites that grow in acidic springs. The top four dominant taxonomic groups found in the TVZ microstromatolites formed in highly acidic hot springs comprised Thermoplasmata, Thermoprotei, Gammaproteobacteria (mainly Acidithiobacillus) and diverse Alphaproteobacteria (Supplementary figure 13). Like other archaea, Thermoplasmata and Thermoprotei have etherlinked lipids which allow them to be more resistant to heat and acidity than bacterial and eukaryotic ester-linked lipids [START_REF] Cavicchioli | Archaea: Molecular and Cellular Biology[END_REF]. This explains why archaea constitute the majority of these very acidic, TVZ hot spring microstromatolite communities. Members of Thermoplasmata and Thermoprotei, along with the bacterium Acidithiobacillus and the Alphaproteobacterium Acidiphilium, have also been identified in acid mine drainage (AMD), where deep within underground mines temperatures can rise up to 30-50°C and pH can drop to negative values [START_REF] Denef | AMD biofilms: using model communities to study microbial evolution and ecological complexity in nature[END_REF][START_REF] Druschel | Acid mine drainage biogeochemistry at Iron Mountain[END_REF].

Other AMD genera, such as Thiomonas, Acidimicrobium and Leptospirillum [START_REF] Baker | Microbial communities in acid mine drainage[END_REF][START_REF] Bond | Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems[END_REF] were detected in digitate sinter from Orange Spring as well as other highly acidic springs in this study. Nevertheless, when we compared our results with non-stromatolite-forming microbial mats from other acidic hot springs elsewhere outside of the TVZ [START_REF] Jackson | Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring[END_REF][START_REF] Macur | Linking geochemical processes with microbial community analysis: successional dynamics in an arsenic-rich, acid-sulfatechloride geothermal spring[END_REF], we found that those mat communities shared little similarity with the TVZ microstromatolite communities from acidic springs.

Cyanidiophyceae dominates microstromatolites in highly acidic springs while diverse eukaryotes characterize higher pH communities

The dominant eukaryotic taxon in highly acidic microstromatolites in the TVZ was Cyanidiophyceae (Archaeplastida, Rhodophyta), a thermoacidophilic, unicellular family of red microalgae that is usually found in volcanic areas or other environments with extremely low pH (0.5-3.0) and temperatures up to 56°C (Fig. 6a, Supplementary figure 14-15) [START_REF] Doemel | The upper temperature limit of Cyanidium caldarium[END_REF][START_REF] Gross | Biogeography of the Cyanidiaceae (Rhodophyta) based on 18S ribosomal RNA sequence data[END_REF]. They were previously reported in sinters along discharge channels at Parariki Stream [START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF], where pH values are low and temperatures are moderate. In addition, they have been observed in many other highly acidic and moderate temperature hot springs, such as at White Island (New Zealand), Seltun (Iceland), Hveradalir (Iceland), and Nymph Creek (YNP), regardless of sample type (e.g. sediment, microbial mat and water) [START_REF] Aguilera | Eukaryotic microbial diversity of phototrophic microbial mats in two Icelandic geothermal hot springs[END_REF][START_REF] Donachie | Microbial community in acidic hydrothermal waters of volcanically active White Island, New Zealand[END_REF][START_REF] Ferris | Algal species and light microenvironment in a low-pH, geothermal microbial mat community[END_REF]. The ability of Cyanidiophyceae to thrive in hot and acidic conditions was conferred by horizontal gene transfer events from both archaea and bacteria [START_REF] Schönknecht | Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote[END_REF]. With a lack of cyanobacteria in highly acidic ecosystems, members of Cyanidiophyceae appear to be the principal photosynthetic organisms (Fig. 6a and 7a; [START_REF] Doemel | The upper temperature limit of Cyanidium caldarium[END_REF].

In circumneutral springs, SAR group (mainly Stramenopiles) dominated more than half of the subaerial TVZ microstromatolite communities (Supplementary figure 1415). Many taxa identified are similar to those found in spring deposits outside the TVZ. For example, eukaryotes associated with TVZ circumneutral spring sinters, such as Bdelloidea (Rotifera), Intramacronucleata (Ciliophora), and Trebouxiophyceae (Chlorophyta), are also associated with a GOPA stromatolite [START_REF] Schuler | Hot spring microbial community composition, morphology, and carbon fixation: Implications for interpreting the ancient rock record[END_REF]. A wide range of other circumneutral digitate sinter taxa (Synurophyceae, Chlorophyta, Cercozoa, Bacillariophyceae, Alveolata and Euglenozoa) are similar to those from non-stromatolitic, microbial mat communities in Icelandic hot springs (pH=6.1-7.4, temperature=15-55°C) [START_REF] Aguilera | Eukaryotic microbial diversity of phototrophic microbial mats in two Icelandic geothermal hot springs[END_REF]. This suggests that sample type (stromatolite vs. microbial mat) does not greatly influence eukaryotic community composition, compared to prokaryotes, in circumneutral hot springs.

Members of Ecdysozoa, particularly Insecta, were very abundant in our alkali-chloride microstromatolite community. They have been identified many times in modern stromatolite records [START_REF] Bertrand-Sarfati | Phanerozoic stromatolites II[END_REF]. Most hot spring insects spend the early stages of their life cycles in the water and their mature stage upon the microbial mats, feeding on mat microorganisms [START_REF] Brock | Feeding by Paracoenia and Ephydra (Diptera> Ephydridae) on the microorganisms of hot springs[END_REF][START_REF] Renaut | Hydrothermal environments, terrestrial. Encyclopedia of Geobiology[END_REF][START_REF] Winterbourn | The distribution of algae and insects in hot spring thermal gradients at Waimangu, New Zealand[END_REF].

Other macroeukaryotes found in the TVZ alkali microstromatolites in this study, such as higher plants, do not live directly in the hot pools, but rather in distal marsh zones or surrounding geothermally influenced environments (Campbell et al., 2015a;Channing & Edwards, 2009a;[START_REF] Channing | Silicification of higher plants in geothermally influenced wetlands: Yellowstone as a Lower Devonian Rhynie analog[END_REF][START_REF] Channing | Wetland megabias: ecological and ecophysiological filtering dominates the fossil record of hot spring floras[END_REF][START_REF] Renaut | Hydrothermal environments, terrestrial. Encyclopedia of Geobiology[END_REF]. Thus, the TVZ siliceous sinter microstromatolites, particularly those formed in the Whangapaoa alkali-chloride spring, not only entomb species that thrive in the springs, but also fossilize biomass transported from surrounding areas or wherever spring fluids infiltrate (cf. [START_REF] Campbell | Late Pleistocene siliceous sinter associated with fluvial, lacustrine, volcaniclastic and landslide deposits at Tahunaatara, Taupo Volcanic Zone, New Zealand[END_REF][START_REF] Trewin | Rhynie Chert[END_REF], providing information on, and preservation of, the wider geothermal environment [START_REF] Walter | Interpreting stromatolites: these fossils can tell us much about past organisms and environments if we can learn to decode their message[END_REF].

Hot spring physicochemistry and sinter environment control species composition

Although it has been shown that salinity is the most important driver of microbial compositional differences in global scale community distribution patterns [START_REF] Lozupone | Global patterns in bacterial diversity[END_REF], our findings suggest a different trend with respect to hot spring communities in the TVZ digitate sinters. When comparing microstromatolite communities from two nearby pools in the same geothermal area with vastly different salinity contents (Pool RTB versus Pool RTG; Supplementary Table 1), no appreciable difference in microbial community composition was observed (Fig. 6a).

We also compared communities from the studied TVZ sites with stromatolite communities from another hypersaline hot spring of similar pH and temperature: a high-altitude hot spring in volcanic Lake Socompa, Argentina (Na = 91 mg/l and Cl = 255 mg/l) (Farais et al., 2013). The Argentine lake communities were shared with the stromatolite communities from TIK3 despite having orders of magnitude difference in salinity content. These results suggest that salinity may not be the most important factor influencing hot spring stromatolite communities. Instead, we found that other physicochemical properties (i.e. the main ions present in water, pH, temperature, and degree of subaerial exposure), were the primary controlling factors of microbial community composition in the studied geothermal spring systems we studied. Abrupt shifts in community composition among TVZ digitate sinters were observed where any one of these drivers changed (Fig. 5, 6a, 7a and 7b). Studies of various other hot spring systems have likewise shown that differences in physicochemical parameters such as these are associated with distinct assemblages of taxa [START_REF] Krebs | Microbial community structures of novel Icelandic hot spring systems revealed by phyloChip G3 analysis[END_REF][START_REF] Nishiyama | The relationship between microbial community structures and environmental parameters revealed by metagenomic analysis of hot spring water in the Kirishima Area, Japan[END_REF][START_REF] Power | Microbial biogeography of 925 geothermal springs in New Zealand[END_REF][START_REF] Renaut | Hydrothermal environments, terrestrial. Encyclopedia of Geobiology[END_REF][START_REF] Sanchez-Garcia | Microbial biomarker transition in high-altitude sinter mounds From El Tatio (Chile) through different stages of hydrothermal activity[END_REF].

Importantly, at the Parariki Stream site (section 3.3.2) we determined that subaerial sinter communities with similar spring physicochemistry were highly similar, regardless of whether the sinter macro-morphology was digitate or otherwise, suggesting that the degree of subaerial exposure (in other words -the degree of thermal water bathing) is another major factor controlling sinter microbial community compositions (Fig. 6b). These results are in line with previous research at Champagne Pool, Wai-O-Tapu, showing differences in bacterial and archaeal community compositions between subaerial and air-water interface sinter communities [START_REF] Childs | Relating microbial community and physicochemical parameters of a hot spring: Champagne Pool, Wai-o-tapu, New Zealand[END_REF]. Moreover, although disparate subaerial sinters were colonized by similar microbial communities at the Parariki Stream locality, the communities perform different roles in sinter build-up. For example, while microbial cells contributed to the formation of micro-lamination in the digitate sinters at this site (Fig. 4b, 4e, 4h and 4k; [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF], in other sinter morphologic types (i.e. cup-shaped and sheet-shaped, section 3.3.2) they appear to contribute only to bulk sinter build-up with no internal micro-laminations [START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF]. As layering in digitate sinters develops due to temporal variations in wetting and drying, which causes fluctuations in silica deposition relative to biofilm development [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF], the lack of laminations in cup and sheet shaped sinters tends to suggest that they develop under comparatively stable conditions. While variation in sinter morphology appears to be controlled by environmental factors, such as the amount of water, gas discharge, spring activity, hydrodynamics and air temperature [START_REF] Braunstein | Relationship between spring and geyser activity and the deposition and morphology of high temperature (> 73 degrees C) siliceous sinter, Yellowstone National[END_REF][START_REF] Nicolau | Physico-chemical and environmental controls on siliceous sinter formation at the high-altitude El Tatio geothermal field, Chile[END_REF][START_REF] Schinteie | Siliceous sinter facies and microbial mats from acid-sulfate-chloride springs, Parariki Stream, Rotokawa geothermal field, Taupo Volcanic Zone[END_REF][START_REF] Schinteie | Microfacies of stromatolitic sinter from acid-sulfate-chloride springs at Parariki Stream, Rotokawa Geothermal Field, New Zealand[END_REF], variation in microbial community composition depends mainly on geothermal fluid characteristics (i.e. temperature, pH, water chemistry, and the degree of subaerial vs subaqueous exposure).

Prokaryotes mostly outnumbered eukaryotes in both diversity and quantity

Because prokaryotes have more efficient mechanisms for coping with extreme conditions compared to eukaryotes [START_REF] Colman | Geobiological feedbacks and the evolution of thermoacidophiles[END_REF], we would predict that prokaryotes are better colonizers of diverse hot spring environments. This would explain why prokaryotic species dominated 9 out of 11 of the hot spring microstromatolite communities we sampled (Fig. 9). Of these, archaea, which have a known preference for extreme conditions [START_REF] Woese | Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya[END_REF], tended to dominate most of the highly acidic spring sinters. This also explains why archaeal diversity increased with temperature and decreasing pH -the opposite trend to that observed for bacteria or eukaryotes. Moreover, an extensive study of the thermal waters of more than 900 hot springs throughout the TVZ showed that, overall prokaryotic diversity was positively correlated with pH [START_REF] Power | Microbial biogeography of 925 geothermal springs in New Zealand[END_REF]. This corresponds well with our microbiology study of actively growing sinters, which shows that prokaryotic (bacterial and archaeal) circumneutral and alkali-chloride microstromatolite communities exhibited higher diversity than the highly acidic ones (Fig. 8). Furthermore, due to the preference of Cyanidiophyceae for highly acidic environments, they dominated the eukaryotic fraction of communities associated with our highly acidic hot spring sinters, resulting in considerably lower observed eukaryotic diversities in these samples.

Future direction and application to Mars

Additional work is needed across a wide range of physicochemical and environmental conditions in order to validate the apparent relationship between morphologic subtypes of digitate sinter and hydrodynamics. In addition, growing more digitate sinters under controlled conditions (e.g. on glass slides for temporal sampling), in conjunction with collecting environmental data, such as weather, water flow or turbulence, and air moisture (cf. [START_REF] Handley | Abiotic-biotic controls on the origin and development of spicular sinter: In situ growth experiments, Champagne Pool, Waiotapu, New Zealand[END_REF], will likely help determine the other factors that control heterogeneity of digitate sinter. If hydrodynamics is confirmed as one of the key factors controlling morphological variation in digitate sinters, then this implies that morphologic subtypes of these textures in extinct hot springs would also be a predictor of paleohydrodynamics, at least where digitate sinter forms. This might also be applicable to the digitate opaline silica at Columbia Hills, Mars.

The physicochemistry of the inferred Martian hot spring deposits is currently unknown, as is the origin of the digitate structures. However, this study shows that digitate sinter can form across a wide range of physicochemical conditions, all of which support the growth of microbial cells. Furthermore, digitate sinter from the studied TVZ sites, as well as El Tatio examples have the potential to fossilize various taxa, and to preserve the size and shape of the varied entombed microorganisms. This implies that if microbial life was once present in hot springs on Mars in the distant past, it potentially could have been preserved and fossilized in the digitate sinter, and generated microbial laminations, which is one of the key textural biosignatures to target in future missions to search for life on the red planet.

Conclusions

New Zealand's digitate sinters form in hot springs with a range of temperatures and pH ranges and are common in shallow outflow channels and around pool rims, where a microbial component is always present. This study explored the relationship among fluid chemistry and hydrodynamics, microorganisms, and the morphology of digitate sinters. We found that the studied TVZ digitate sinter microstromatolites are universally laminated, and host and silicify a diverse array of microorganisms. However, microbial community composition was not correlated with digitate sinter morphologic subtypes in the subaerial environment. Instead, results suggest that hydrodynamics is at least one of the factors influencing digitate sinter morphological variation. The primary controllers of the composition of TVZ digitate sinter microbial communities were water chemistry, pH, temperature, and degree of subaerial exposure. In line with this conclusion, we found that TVZ microstromatolites share microbial taxa with stromatolites grown in physicochemically similar hot springs elsewhere. We also determined that the diversity and abundance of prokaryotes associated with the studied TVZ digitate sinters is typically much higher than that of eukaryotes. Our finding, that stromatolitic digitate sinters form under a wide range of geothermal fluid conditions, increases the likelihood that they could have formed on early Mars if it ever hosted microbial life.

Figure 1 .

 1 Figure 1. Map of Taupo Volcanic Zone (TVZ) geothermal areas, North Island, New Zealand.

Figure 2 .

 2 Figure 2. Temperature, pH and water chemistry data for the studied hot springs from which each

Figure 3 .

 3 Figure 3. Representative in situ, actively growing digitate sinter from (a-b) an AS spring (Te Kopia

Figure 4 .

 4 Figure 4. SEM photos of digitate sinter samples showing macrostructure, microbial laminations

Figure 8 .

 8 Figure 8. Shannon's index of digitate sinter communities showing (a) prokaryotic diversity and

Figure 9 .

 9 Figure 9. Plot illustrating quantification of prokaryotes and eukaryotes by ddPCR, and showing

Acknowledgements

The authors thank B. Drake, M. Rowe, M. Dobson, S. Camp, Y. Heled, D. P. Aparicio, A. Hamilton and the rest of the members of the Hot Spring Research Group at the University of Auckland (UoA) for facilitating and helping with the field work and geochemistry data collection. We also thank C.

Hobbis (Research

Centre for Surface and Materials Science), A. Turner (School of Biological Sciences) and L. Cotterall (School of Environment) at UoA for technical support. We are grateful to D. Waite, J. Boey and E. Gios from the School of Biological Sciences at UoA for bioinformatics support. We acknowledge Timberlands Ltd (Rotorua) for Orange Spring site access, Mercury Energy (Rotorua) and the New Zealand Department of Conservation (DOC) for Parariki Stream and Lake Rotokawa site access, R. and B. McNaull for Te Kopia site access, Hell's Gate Ltd for Tikitere site access, and DOC for Whangapaoa Spring site access. MVK was supported by the Australian Research Council through grant DP180103204. Finally, the authors wish to acknowledge the use of New Zealand eScience Infrastructure (NeSI) high performance computing facilities.

Funding information

Funding was provided by the University of Auckland Faculty Research Development Fund (grant number 3712371), and a Royal Society Te Apārangi Rutherford Discovery Fellowship awarded to KMH.

Conflict of Interests

The authors declare that they have no conflict of interests.

spring (from pool PA: pH = 1.0, temperature = 49.0°C, and pool RTSH: pH = 1.73, temperature = 53.0°C); (g-i) an ASB spring (from pool TIK2: pH = 5.9, temperature = 38.9°C, and pool TIK3: pH = 6.6, temperature = 34.5°C); and (j-l) an AC spring (from pool WH: pH = 8.5, temperature = 48.1°C).


Emmanuel Bresso

Pierre Monnin

Cédric Bousquet

François-Élie Calvier

Ndeye Coumba Ndiaye

Nadine Petitpain

Malika Smaïl-Tabbone

Adrien Coulet

Investigating ADR mechanisms with Explainable AI: a feasibility study with knowledge graph mining

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Investigating ADR mechanisms with Explainable AI: a feasibility study with knowledge graph mining Emmanuel Bresso 1,2 , Pierre Monnin 1,2 , Cédric Bousquet 2 , François-Elie Calvier 2 , Ndeye-Coumba Ndiaye 2 , Nadine Petitpain 2 , Malika Smaïl-Tabbone 1 and Adrien Coulet 1,2 Adverse drug reactions (ADRs) are statistically characterized within randomized trials or by postmarketing pharmacovigilance. However, the molecular mechanisms causing ADRs remain unknown in most cases. This is true even for common toxicities that are classically monitored during trials such as hepatic or skin toxicities. Interestingly, many elements of knowledge about drugs and drug ingredients are available beside clinical trials. In particular, open-access knowledge graphs describe their properties, interactions, and involvements in pathways. Expert classifications have also been manually established by experts and label drugs either as causative or not for several types of ADRs.

In our paper [START_REF] Bresso | Investigating ADR mechanisms with explainable AI: a feasibility study with knowledge graph mining[END_REF], we propose to mine biomedical knowledge graphs to identify biomolecular features that enable to automatically reproduce such expert classifications, distinguishing drugs causative or not for a given type of ADR. In an Explainable AI perspective, we explore simple classification techniques such as Decision Trees and Classification Rules because they provide human-readable models which explain the classification itself. We also evaluate the assumption that biomolecular features mined from knowledge graphs might provide elements of explanation for the molecular mechanisms behind ADRs. We tested our approach with two expert classifications identifying drugs causing or not hepatic or skin toxicities (respectively named DILI and SCAR for Drug Induced Liver Injuries and Severe Cutaneous Adverse Reactions). Features associated with these drugs were mined from PGxLOD [START_REF] Monnin | PGxO and PGxLOD: a reconciliation of pharmacogenomic knowledge of various provenances, enabling further comparison[END_REF], a biomedical knowledge graph that we previously created by interlinking public open data (including DisGeNET, PharmGKB, DrugBank, CTD). To this aim, we developed the kgpm algorithm [START_REF] Monnin | Tackling scalability issues in mining path patterns from knowledge graphs: a preliminary study[END_REF] that enables scaling the extraction of paths of features up to a length of 4. Such paths are subsequently generalized into path patterns to cover larger sets of drugs. We trained two classifiers at distinguishing, on the basis of extracted features, drugs causing or not each of the two considered ADRs. We isolated features that are both discriminative in reproducing expert classifications and interpretable by experts (e.g., Gene Ontology terms, drug targets, pathways) and asked 3 pharmacology experts to manually evaluate if they are potentially explanatory for ADRs.

Extracted features reproduce DILI and SCAR classifications with a fair fidelity (accuracy of 0.74 and 0.81, respectively). Experts fully agreed that 73% and 38% of the most discriminative features are possibly explanatory for DILI and SCAR, respectively; and partially agreed (2/3) for 90% and 77% of them. To illustrate, the path pattern interactsWith --------→ Enzyme cellularComponent -----------→ Endoplasmic reticulum reached an agreement for DILI. Indeed, the endoplasmic reticulum is known, in particular in liver tissues, to host primarily cytochrome P450 enzymes, well known for being involved in drug metabolism.

From these results, it appears that the considered knowledge graph provides sufficiently diverse features to enable simple and explainable models to distinguish between drugs that are causative or not for ADRs. In addition to explaining expert classifications, most discriminative features appear to be good candidates for investigating further molecular mechanisms behind ADRs.

1

  Université de Lorraine, CNRS, Inria, LORIA, Nancy, France 2 Please see[START_REF] Bresso | Investigating ADR mechanisms with explainable AI: a feasibility study with knowledge graph mining[END_REF] for full author information

	Corresponding author: adrien.coulet@inria.fr
	Reference paper: Bresso et al. (2021) Investigating ADR mechanisms with Explainable AI: a
	feasibility study with knowledge graph mining. BMC Medical Informatics and Decision Making.
	https://doi.org/10.1186/s12911-021-01518-6