Lough Hyne, Ireland) 3

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.290791; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The effects of seasonal anoxia on the microbial community structure in 2 demosponges in a marine lake (Lough Hyne, Ireland) 3 4 Astrid Schuster,a,b#*, Brian William Strehlowa*, Lisa Eckford-Sopera, Rob McAllenc, Donald Eugene 5 Canfielda 6 7 a Department of Biology, University of Southern Denmark, Odense M, Denmark. 8 b Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Matosinhos, 9 Portugal. 10 c School of Biological, Earth and Environmental Sciences, University College Cork, Cork, Ireland. 11 12 Running Head: Stable sponge microbiomes at varied oxygen levels 13 14 #Address correspondence to Astrid Schuster, [email protected] 15 Department of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, 16 Odense M, Denmark. 17 18 *Astrid Schuster and Brian William Strehlow contributed equally to this work. Author order was 19 determined alphabetically on the basis of the last name. 20 21 22 23 24 25 26 27 28 29 30 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.290791; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 31 32 Abstract 33 Climate change is expanding marine oxygen minimum zones (OMZs), while anthropogenic nutrient 34 input depletes oxygen concentrations locally. The effects of deoxygenation on animals are generally 35 detrimental; however, some sponges (Porifera) exhibit hypoxic and anoxic tolerance through 36 currently unknown mechanisms. Sponges harbor highly specific microbiomes, which can include 37 microbes with anaerobic capabilities. Sponge-microbe symbioses must also have persisted through 38 multiple anoxic/hypoxic periods throughout Earth history. Since sponges lack key components of the 39 hypoxia-inducible factor (HIF) pathway responsible for hypoxic responses in other animals, it was 40 hypothesized that sponge tolerance to deoxygenation may be facilitated by its microbiome. To test 41 this hypothesis, we determined the microbial composition of sponge species tolerating seasonal 42 anoxia and hypoxia in situ in a semi-enclosed marine lake, using 16S rRNA amplicon sequencing. We 43 discovered a high degree of cryptic diversity among sponge species tolerating seasonal 44 deoxygenation, including at least nine encrusting species of the orders Axinellida and 45 Poecilosclerida. Despite significant changes in microbial community structure in the water, sponge 46 microbiomes were species specific and remarkably stable under varied oxygen conditions, though 47 some symbiont sharing occurred under anoxia. At least three symbiont combinations, all including 48 large populations of Thaumarchaeota, corresponded with deoxygenation tolerance, and some 49 combinations were shared between distantly related hosts. We propose hypothetical host-symbiont 50 interactions following deoxygenation that could confer deoxygenation tolerance. 51 52 53 54 55 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.290791; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 56 57 58 Importance 59 The oceans have an uncertain future due to anthropogenic stressors and an uncertain past that is 60 becoming clearer with advances in biogeochemistry. Both past and future oceans were, or will be, 61 deoxygenated compared to present conditions. Studying how sponges and their associated 62 microbes tolerate deoxygenation provides insights into future marine ecosystems. Moreover, 63 sponges form the earliest branch of the animal evolutionary tree and they likely resemble some of 64 the first animals. We determined the effects of variable environmental oxygen concentrations on 65 the microbial communities of several demosponge species during seasonal anoxia in the field. Our 66 results indicate that anoxic tolerance in some sponges may depend on their symbionts, but anoxic 67 tolerance was not universal in sponges. Therefore, some sponge species could likely outcompete 68 benthic organisms like corals in future, reduced-oxygen ecosystems. Our results support the 69 molecular evidence that sponges and other animals have a Neoproterozoic origin, and that animal 70 evolution was not limited by low-oxygen conditions. 71 72 73 74 75 76 77 78 79 80 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.290791; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 Introduction 82 Ocean anoxia and hypoxia have become major stressors for many marine organisms. Indeed, 83 oxygen-minimum zones (OMZs) and coastal hypoxic areas will most likely expand in the future (1–5), 84 leading to habitat and biodiversity losses (5–8), where oxygen depletion is caused by both natural 85 and anthropogenic influences (9, 10). While motile species can escape such hypoxic/anoxic areas, 86 many sessile organisms like sponges (Porifera) and corals (Cnidaria) must either cope with these 87 extremes or suffer mass mortalities as observed in Tropical dead zones (11). Nevertheless, the lethal 88 thresholds of, and potential adaptations to, deoxygenation are understudied in these organisms 89 (12), particularly in sponges. 90 Sponges are common, cosmopolitan filter feeders that pump water through their bodies to filter 91 and ingest nutrients and microorganisms (13, 14). Previous ex situ experiments have shown that 92 sponges, including Geodia barretti (15), Tethya wilhelma (16), Halichondria panicea (17), Haliclona 93 pigmentifera (18) and Vazella pourtalesii (19), have a high tolerance of hypoxia. For instance, T. 94 wilhelma can maintain normal transcription at ~0.5 µM O2 (16), despite lacking key components of 95 the hypoxia-inducible factor (HIF) pathway, which regulates hypoxic responses in other 96 invertebrates (16). However, T. wilhelma, H. panacea, and H. pigmentifera became stressed, 97 sometimes fatally, during anoxia (16–18). Some marine sponge species also tolerate hypoxia and 98 even anoxia in their natural environment (20–24), and gemmules from freshwater sponges can 99 survive months of anoxia (25). Nevertheless, hypoxia-induced mortality was reported in situ for the 100 demosponges Aaptos simplex and Homaxinella amphispicula (21), so responses are likely species- 101 specific. The current global distribution and depth range of sponges (26) overlaps with that of 102 hypoxic areas worldwide (2), indicating that many sponges may even thrive in hypoxic 103 environments, perhaps due to limited competition. Therefore, some sponges have likely developed 104 alternative adaptation strategies to tolerate variable and low-oxygen conditions independent of the 105 HIF pathway. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.290791; this version posted September 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 106 Sponges can harbor stable and sometimes diverse microbial communities that can constitute up to 107 50% of their biomass (27, 28); together, the host and microbiome are referred to as the sponge 108 ‘holobiont’ (29). Sponge holobionts play a key role in marine ecosystems, contributing to reef 109 formation, benthic-pelagic nutrient coupling, and biogeochemical cycling (30–32). Some of these 110 sponge-microbe associations are highly stable under environmental stressors, including elevated 111 temperature (33–35), eutrophication and sedimentation (36), and some symbionts are sponge- 112 specific, meaning they occur negligibly in the environment (37). Stable sponge-microbe associations 113 were also found across large geographic distances (38). However, microbial communities across 114 sponge species substantially vary in diversity, structure and abundance (39), and there can be 115 selection for divergent microbiomes, even among related sponge lineages (40). 116 Within a specific holobiont, symbiotic microbes may increase sponge fitness by providing food 117 (through carbon fixation or direct ingestion of symbionts), recycling of nutrients and waste products, 118 and/or the production of secondary metabolites for predator defense or other functions (30, 31). 119 Many sponge species form symbioses with Nitrosopumilus-like ammonia-oxidizing Archaea (AOA) 120 and/or Nitrospira sp. nitrite-oxidizing Bacteria (NOB) (19, 40–44). Each of these prokaryotic groups 121 utilize sponge waste ammonia for nitrification. Similar microbes are active in hypoxic waters and 122 contribute to biogeochemical cycling processes (45). For example, Nitrospira sp. accounted for 9% of 123 the total microbial community in the OMZ of the Benguela upwelling system (46). Also, 124 Nitrosopumilacea sp., a widespread and dominant AOA in many OMZs, plays a significant role in 125 ammonium oxidation therein (47, 48). Thus, many of these common sponge symbionts could be 126 adapted to hypoxia, and it was suggested that the presence of AOA symbionts, and other facultative 127 anaerobes,

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