Journal Section: Invertebrate Microbiology Running Title: Seasonal stability of sponge-bacteria symbioses Stability of sponge- bacteria o ver large seasonal shifts in temperature and irradiance

Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the NW Mediterranean Sea, using replicated terminalrestriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host-species specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living versus symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitriteoxidation and sulfate-reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health. INTRODUCTION Sponges are sessile invertebrates that form a species rich phylum at the base of the metazoan tree of life (>8,500 valid species; 65). Renowned for their efficient filter-feeding capabilities and bioactive secondary metabolite production, sponges have important ecological and biotechnological relevance as major players in marine nutrient cycles (11,12,26) and the most prolific producers of marine natural products (>6,600 secondary metabolites; 16). The discovery and characterization of diverse microbial symbionts inhabiting the sponge body has prompted the adoption of the holobiont concept, thereby incorporating microbial symbionts in the study of sponge ecology and evolution (55). The resulting field of sponge microbiology has grown rapidly in the past two decades (59) and revealed a tight ecological link between host health and symbiont composition. Indeed, sponge-associated microbes have been implicated in host metabolism and growth (20,22,75), chemical defense production (21) and susceptibility to biotic (e.g., disease) and abiotic (e.g., temperature stress) stressors (33,66,72). The remarkable diversity of the sponge microbiota has presented a formidable challenge to understanding the structure and function of microbial guilds in sponge hosts (59,70). The sponge microbiota includes diverse phylogenetic lineages of Archaea and Bacteria, as well as fungi and viruses (52,56). Among bacterial symbionts alone, thousands of taxa have been reported, spanning 17 described phyla and 12 candidate phyla (50), and hundreds of bacterial taxa can occur in a single host individual (32,71). Accordingly, considerable effort has focused on describing the vast diversity of the sponge microbiota, while more applied studies of symbiont functioning have targeted specific components (e.g., Cyanobacteria; 59) or functional gene pathways (e.g., ammonia oxidation; 35) in these communities. As a result, most studies of sponge microbiology have been limited in scope to one or few host species collected at a single time point, thus much of our knowledge concerning the sponge microbiota is based on a static representation of these potentially dynamic communities (59). Understanding the complex sponge microbiota requires a basic knowledge of how these communities change over time. The general consensus is that sponge-microbe associations are largely stable over temporal scales (56), including epibionts (31), cultivatable symbionts (68) and entire bacterial communities (57,60,61,73). Other studies have reported higher levels of variability across seasons (74) and when repeatedly sampling the same individuals over time (3), indicating some degree of symbiont fluctuation over time and individual variation among hosts. The prospect of sponge aquaculture for the production of bioactive metabolites has prompted investigations of host-symbiont stability under ex situ aquaria conditions, revealing high symbiont stability over short-term time scales (11 days to 12 weeks; 23,67), while longer-term maintenance (six months to two years) can result in substantial shifts in symbiont composition (39,40,67). Additional studies of temporal variation in sponge-associated bacteria under natural conditions will aid future aquaculture efforts by determining natural variation in the sponge microbiota and its consequences for host-symbiont dynamics. Further, such studies establish the baseline levels of symbiont variability required to define abnormal shifts and ascribe symbiont fluctuations to specific abiotic and biotic factors. In this study, we investigated temporal variation in the microbiota of three congeneric sponge hosts from the Mediterranean Sea: Ircinia fasciculata, I. variabilis and I. oros. These sponges are common members of coastal benthic communities in the Mediterranean Sea and harbor diverse, host-specific communities of bacterial and cyanobacterial symbionts (15,17,46, 63). Replicate individuals of each sponge species were tagged in situ and sampled quarterly for 1.5 years to monitor their bacterial symbiont communities, using terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S ribosomal RNA (rRNA) gene sequences. In addition, photosynthetic pigments were monitored in the tissues of the cyanobacteria-rich sponges I. fasciculata and I. variabilis, using chlorophyll a quantification. The specific objectives of the study were: 1) determine the temporal stability of host-symbiont specificity, 2) identify permanent and transient symbiont taxa in association with sponge hosts, and 3) document natural variability in symbiont communities over time. Collectively, these objectives contribute to the broader goal of establishing the empirical baselines required to diagnose abnormal symbiont shifts and develop these symbiotic systems as an impact assessment tool in coastal ecosystems. METHODS Sample collection The sponge species Ircinia fasciculata (PALLAS 1766), I. variabilis (SCHMIDT, 1862) and I. oros (SCHMIDT, 1864) were monitored in shallow (< 20 m) littoral zones at two neighboring sites (< 12 km apart) along the Catalan Coast (Spain) in the northwestern Mediterranean Sea. I. fasciculata colonies were studied in Punta de S’Agulla (Blanes; 41o 40’ 54.87” N, 2o 49’ 00.01” E) and I. variabilis and I. oros in Mar Menuda (Tossa de Mar; 41o 43’ 13.62” N, 2o 56’ 26.90” E) from March 2010 to June 2011. Initial sampling of I. oros (March 2010) was performed in the nearby Punta Santa Anna (Blanes; 41o 40’ 21.48” N, 2o 48’ 13.55” E); however, from June 2010 to June 2011, sampling was conducted in Tossa de Mar, due to the onset of heavy construction in the adjacent Blanes Port (< 300 m from the Punta Santa Anna sampling site) in May 2010. Individual sponges were marked in situ and sampled quarterly for genetic analyses and chlorophyll a concentrations by SCUBA diving using a scalpel blade and forceps. At each site, ambient seawater samples (500 ml) were collected simultaneously and in close proximity (< 1 m) to sampled sponges. Sponge and seawater samples were transported in an insulated cooler to the laboratory (ca. two hrs. transit time), where sponge samples for genetic analyses were preserved in 100% ethanol and stored at -20oC and seawater samples were concentrated on 0.2 μm filters and stored at -80oC. Tissue samples for chlorophyll a quantification were processed immediately