*3.1. Core Microbiome*

Ascidian (host)-microbiome specificity is an active area of research. Compared to sponges and corals, for example, ascidian microbiomes have been less well characterized. To achieve a broad perspective on the microbiome composition, we also used cultivation-independent approaches. We found that the Antarctic ascidian *S. adareanum* has a persistent core microbiome across the Anvers Island Archipelago that is distinct from the plankton. This dissimilarity between ascidian host-associated microorganisms and bacterioplankton appears to be a consistent observation across the global ocean (e.g., [16–19]). The Core80 is comprised of ASVs that numerically dominate the community, as well as those representing only a fraction of a percent of the sequences surveyed. Although ascidian symbioses have not ye<sup>t</sup> been systematically studied in the Antarctic, better-studied lower latitude ascidian microbiomes provide several examples for comparison. The overall trend across ascidian microbiome studies to date suggests that there is a high degree of both geographical as well as host species-level specificity of microbiome composition (e.g., [16,17,20]). The same appears to be true of *S. adareanum*. Although this study was restricted to a small geographical region, we identified a conserved core of 21 16S rRNA gene sequence types across 63 individual pedunculate lobes studied. We attribute the detection of this high degree of persistent members in part to the uniform homogenization, extraction, and sequencing methodological pipeline applied. Microbiome analysis is sensitive to sequencing depth, quality parameter choices, and algorithmic di fferences in data-processing pipelines (amplicon sequence variants vs. cluster-derived operational taxonomic units), which can impact direct comparisons between studies. Along these lines, the numerous highly related *Microbulbifer* ASVs would have fallen into a single OTU (97% sequence identity), resulting in a core with 14 members. These limitations aside, our findings are in line with several other ascidian microbiome studies from lower latitudes in terms of the relative size of core membership (where core definitions vary to some degree between studies). For example, *Styela plicata*, a solitary ascidian, was reported to have a core membership of 10 [21] to 16 OTUs [22]. Other solitary ascidians, including *Herdmania momus* had a core of 17 OTUs [21], while two *Ciona* species ranged from 8 to 9 OTUs [23]. Temperate colonial ascidians *Botryliodes leachi* and *Botryllus schlosseri* ranged from 10 to 11 members in their core microbiomes [20]. Further, an extensive survey of 10 di fferent ascidian microbiomes (representing both solitary and colonial forms) conducted on the Great Barrier Reef reported core memberships ranging from 2 to 35 OTUs [16], while the numbers of individuals surveyed in each case were only 2–3. Note that a few other studies reported much higher numbers of shared OTUs ranging from 93 to 238 [18,19]; the scale of sequencing was higher in these later studies. Further, as others have reported [24], the membership of these core ascidian microbiomes is distinct, and in the case of SaM, the core microbiome diversity appears to be unique at the ASV level, although several taxa are in common with other ascidian-associated microbes at the genus level including *Microbulbifer* associated with *Cystodytes* sp. [25], *Pseudovibrio* with *Polycitor proliferus* [26] and an *Endozoicomonas*-specific clade was identified in a survey of a number of ascidians [27].

Predicted metabolic abilities of the Core80 taxa sugges<sup>t</sup> aerobic heterotrophy (aerobic respiration—organic carbon is the carbon and energy source), microaerophily (growth in low oxygen conditions) and chemoautotrophy (CO2 fixation provides carbon and reduced chemicals provide energy, e.g., NH4 + and NO2 −) are themes amongs<sup>t</sup> the Core80, in which the most abundant ASVs are high-molecular-weight carbon degraders. The *Microbulbifer* genus has members known to degrade cellulose [28], and perhaps noncoincidently, ascidians are the only known invertebrate capable of cellulose biosynthesis in the marine environment (e.g., [29,30]). From this, we could speculate that the *Microbulbifer* strains associated with *S. adareanum* could occupy a commensal, if not somewhat antagonistic, relationship [31]. In support of this possibility is the fact that the only overlapping sequence between the Core80 and the bacterioplankton was a *Microbulbifer* sequence, which was a rare sequence in the plankton, suggesting that it may be an opportunistic member of the *S. adareanum* microbiome. In addition, free-living and sponge-associated isolates from the *Microbulbifer* genus have been found to produce bioactive compounds including pelagiomicins [32] and parabens [33], respectively. This observation, in the least, suggests that the *Microbulbifer(s)* is/are likely well adapted to their ascidian host and might be considered a potential PalA-producing organism.

The NH4 +-oxidizing *Nitrosopumilis*-a ffiliated Thaumarchaeota have been commonly detected in ascidian microbiomes [16,21,22,34], which contrasts phylogenetically, but not in terms of biogeochemical function, with the NH4 +-oxidizing *Nitrosomonas* ASV that was part of the SaM core. The niche, however, has been reported to be di fferent for the archaeal and bacterial NH4 + oxidizers, in which the archaea tend to be found in oligotrophic systems, while the bacteria (e.g., *Nitrosomonas*) can tolerate high levels of dissolved ammonia (reviewed by [35]). This result might reflect both the environment, and in situ *S. adareanum* tissue ammonia levels where it may accumulate. Several studies have reported on the high levels of oxidizing ammonia Thaumarchaeota in the coastal waters of the Anvers Island Archipelago which are numerous only in winter to early spring waters [36–38]. Our study was conducted with samples collected in Fall, when the ammonia-oxidizing Thaumarchaeota are not abundant in the coastal seawater [36], advocating for the comparisons between the SaM with bacterioplankton collected in both late summer and winter periods.

In a similar vein, although we did not intentionally conduct a temporal study, the data from samples collected in 2007 and 2011 appear to sugges<sup>t</sup> that a number of the core microorganisms are stable over time. We found several ASVs in 2011 samples that matched (at 100% sequence identity) cloned sequences from samples collected in 2007 [13]. Stability of the ascidian microbiome over time has been reported in a few studies [17,24,34]. Studying the persistence of the core membership over the annual cycle would be interesting (and provide compelling evidence for stable relationships) in this high-latitude environment, where light, carbon production, and sea ice cover are highly variable.

The co-occurrence analysis indicated three subsystems of ASVs that co-occur within *S. adareanum*. A small side network included the two taxa involved with the 2-step nitrification process, including the *Nitrosomonas* ASV mentioned above and a *Nitrospira* ASV. Even though at present, the functional underpinnings of the host–microbial system have not been studied, the co-occurrence relationships provide fodder for hypothesis testing in the future. One interaction network that warrants mentioning here is the ASVs in Subsystem 1, which harbor several Core80 *Microbulbifer* ASVs, and the *Pseudovibrio* ASV are linked to a *Bdellovibrio* ASV that is also a member of the Core80. Members of the *Bdellovibrio* genus are obligate bacterial predators [39] that penetrate the outer membrane and cell wall of their prey. The linkage position in the subsystem is compelling in the sense that the *Bdellovibrio* could potentially control the abundance of the connected members of the network. Lastly, the positions of a couple of Dynamic and several the Variable ASVs in the network, as links between the subsystems, was unexpected. The central positions of these ASVs sugges<sup>t</sup> that they may not be merely stochastic members of the microbiome; that they could play opportunistic, adaptive or ecological roles in the functionality of the microbiome subsystem(s) which potentially participate in di fferent aspects of the holobiont system in particular, by promoting the switch between di fferent ecological modes supported by di fferent subsystems. Such roles were proposed for dynamic members of the *Styela plicata* microbiome [21].

The culturing e ffort succeeded in isolating a *Pseudovibrio* strain that is a crucial member of the Core80. In addition, several other Gammaproteobacteria-a ffiliated strains which matched sequences in the Variable SaM and the bacterioplankton were cultivated. However, the cultivated diversity using the approaches applied here reared a collection of limited diversity. It is likely that additional media types and isolation strategies could result in additional cultivated diversity as there are a number of taxa with aerobic heterotrophic lifestyles in the Core80 that have been brought into pure culture (e.g., *Microbulbifer*, *Hoeflea*). One challenge we experienced using the nutrient-replete media was overgrowth of plates, even at 10 ◦C.

### *3.2. Secondary Metabolite Distributions and Bioaccumulation in Marine Biota*

Although the results of the archipelago spatial ascidian survey did not support a direct relationship between PalA levels and the relative abundance of microbiome ASVs, the results of the PalA niche analysis suggests that the Core80 ASVs occur in a preferred optimum and tolerance range of PalA levels. The lack of specific ASV-PalA patterns may not be entirely surprising, as secondary metabolites result from a complex combination of metabolic reactions that require a fine-tuning to environmental conditions and further metabolic modeling for the sake of understanding. Furthermore, these metabolites have been found to accumulate in the tissues in several di fferent marine invertebrates. The Optimal Defense Theory can be applied to marine invertebrates and reflects the hypothesis that secondary metabolites are distributed in specific tissues based on exposure and anatomic susceptibility for predation [39]. For example, nudibranchs sequester toxic compounds, which have been biosynthesized by the gastropod or acquired from their prey. The toxins are concentrated in the anatomical space of their mantles, the most vulnerable portion of their soft, exposed bodies [40–42]. Bioaccumulation of secondary metabolites in invertebrates with less anatomical di fferentiation is also known to occur. In the phylum Porifera, di fferent cell types and layers have been studied to determine spatial and anatomical di fferences in secondary metabolite concentrations [43–45]. Compounds have been found to be concentrated spatially on the surface (e.g., [46]) or apical parts of the sponge [47] in some cases. Sponges may be able to di fferentially bioaccumulate secondary cytotoxic metabolites based on tissues more susceptible to predation [48]. Metabolite distribution investigations that are ascidian-specific are less well documented; however, there is also evidence of ascidian secondary cytotoxic metabolite bioaccumulation. The patellazoles, marine macrolides from the ascidian *Lissoclinum patella*, bioaccumulate in the ascidian tissues to concentrations up to seven orders of magnitude higher than their cytotoxic dose in mammalian cell lines [49,50]. Additionally, there are other instances in which bioaccumulation in ascidian host tissues suggests metabolic cooperation of producer and host as well as compound translocation from producer to host [15,51,52]. Although the PalA levels were normalized to grams of dry lobe weight, tissue-specific spatial localization is a potentially confounding factor in the statistical analyses investigating the ASV:PalA relationship.

### *3.3. The Biosynthetic Potential of the Core*

We investigated the natural product biosynthetic potential of the nine genera associated with 15 of 21 Core80 ASVs using antiSMASH (Table 2, Table S4). From this, it appears that all genera had at least one relative at the genus level with biosynthetic capacity for either polyketide or nonribosomal peptide biosynthesis or both. Even though the number of genomes available to survey were highly uneven, there is quite a disparity of biosynthetic capacity between the genera analyzed, thus, it appears that *Pseudovibrio*, *Nitrosomonas*, *Microbulbifer*, *Nitrospira* have the greatest capacities (in that order). Likewise, *Microbulbifer*, *Pseudovibrio*, *Hoeflea* and *Opitutaceae* might be prioritized as candidate PalA producers based solely on relative abundance ranking (Table 2; [24]). Although we did not conduct this analysis for the six ASVs that were classified at best at the family or order level, a few of these might be worth considering as potential producers considering their higher-level relationships with marine natural product producing lineages. For example, marine actinobacteria are classically associated with the production of numerous bioactive natural products (e.g., [53,54]), although speculation is di fficult with actinobacteria SaM\_ASV20 in the core as it is only distantly related to known natural product producers. Likewise, *Opitutaceae*-related SaM\_ASV15 is ranked 7 in terms of average relative abundance and falls in the same family the ascidian-associated *Candidatus* Didemnitutus mandela, which harbors the biosynthetic gene cluster predicted to produce mandelalide, a glycosylated polyketide [55]. From this, we might prioritize the *Microbulbifer*, *Pseudovibrio*, and *Opitutaceae* ASVs for downstream investigation, with the lower relative abundance *Nitrosomonas* and *Nitrospira* ASVs also holding some potential given the perhaps surprising abundance of biosynthetic gene cluster content in these chemoautotrophic, and generally small genome-size taxa. Although this analysis focused on predicted pathway characterizations across genera detected, the distributions of predicted pathways varied substantially across the taxa analyzed. The potential for these new Antarctic ascidian-associated strains to harbor secondary metabolite pathways remains speculative as they are amongs<sup>t</sup> the most variable component of a bacterium's genome.

**Table 2.** Taxonomic a ffiliations of core microbiome, relative abundance rank, and the potential of affiliated genus in natural product gene cluster biosynthesis. Taxonomy is shown according to genome taxonomy database (GTDB) classification and NCBI taxonomy is included (GTDB/NCBI) where they di ffer. Biosynthetic potential only calculated for ASVs with genus-level taxonomic assignments was based on representative genome biosynthetic gene cluster content in the same genus (See Figure S3 for list of genomes). ASVs in bold ranked in the top 10. Where more than one ASV was found per genus, the average relative abundance and standard deviations were summed. n = 63 individuals.

