**1. Introduction**

Microbial partners of marine invertebrates play intrinsic roles in the marine environment at both the individual (host survival) and community (species distribution) levels. Host–microbe relationships are mediated through complex interactions that can include nutrient exchange, environmental adaptation, and production of defensive metabolites. These functional interactions are tied to the structural nature (diversity, biogeography, and stability) of host and microbiome, and the ecological interactions between

them. Studies of sponges, corals, and, to a lesser degree, ascidians have revealed strong trends in invertebrate host species specificity to particular groups of bacteria and archaea. These studies have documented an underlying layer of diversity (e.g., [1–3]) in which habitat and biogeography appear to have strong influences on the microbiome structure and function [4–6].

The vast majority of host–microbiome studies have been conducted at low- and mid-latitudes from coastal to deep-sea sites. High-latitude benthic marine invertebrate-associated microbiome studies are currently limited to the Antarctic, where just the tip of the iceberg has been investigated in regards to di fferent host–microbe associations [7] and ecological understanding is sparse. Antarctic marine invertebrates tend to have a high degree of endemicity at the species level, often display circumpolar distribution, and in many cases have closest relatives associated with deep-sea fauna. Whether endemicity dominates the microbiomes of high-latitude benthic invertebrate is currently not known, nor is the extent of diversity understood within and between di fferent host-associated microbiomes. Likewise, reports of core (conserved within a host species) microbiomes within Antarctic invertebrate species are sparse.

The few polar host-associated microbiome studies to date have documented varying trends in host species specificity, with generally low numbers of individuals surveyed. For example, low species specificity was reported in sponge microbiome compositions between di fferent sub-Antarctic and South Shetland Island *Mycale* species [8] which shared 74% of the operational taxonomic units (OTUs), possibly representing a cross-*Mycale* core microbiome. On the contrary, high levels of microbiome–host species specificity and shared core sequences within a species was observed in five McMurdo Sound sponge species [9]. The same was found across several Antarctic continental shelf sponge species [10]. Webster and Bourne [11] also found conserved bacterial taxa dominated by microorganisms in the class Gammaproteobacteria across the soft coral, *Alcyonium antarcticum*, sampled at three sites in McMurdo Sound. Another cnidarian, the novel ice shelf burrowing sea anemone *Edswardsiella andrillae*, contained novel microbiota, though the composition across a limited set of individuals was only moderately conserved, in which some specimens were dominated by an OTU associated with the phylum Tenericutes, and others, a novel OTU in the class Alphaproteobacteria [12]. Lastly, a single representative of the Antarctic ascidian *Synoicum adareanum* revealed limited rRNA gene sequence diversity, including representatives of Actinobacteria, Bacteroidetes, Proteobacteria, Verrucomicrobia, and TM7 phyla [13], though persistence of these taxa across individuals was not studied.

*Alcyonium antarcticum* (formerly, *A. paessleri*) and *Synoicum adareanum* are both reported to be rich in secondary metabolites. The soft coral *A. antarcticum* produces sesquiterpenes that are unusual in bearing nitrate ester functional groups [14], while the ascidian, *S. adareanum,* is known to produce a family of macrolide polyketides, the palmerolides, which have potent activity against melanoma [15]. The role of the microbial community in contributing to host defensive chemistry, microbe–chemistry interactions and niche optimization, as well as microbe–microbe interactions, are unknown in these high-latitude environments.

Here, we have designed a study to investigate whether a core microbiome persists among *S. adareanum* holobionts that may inform our understanding of palmerolide origins. We conducted a spatial survey of *S. adareanum* in which we studied coordinated specimen-level quantitation of the major secondary metabolite, palmerolide A (PalA), along with the host-associated microbiome diversity and community structure across the Anvers Island Archipelago (64◦46- S, 64◦03- W) on the Antarctic Peninsula (Figure 1). The results point to a core suite of microbes associated with PalA-containing *S. adareanum*, distinct from the bacterioplankton, which will lead to downstream testing of the hypothesis that the PalA producer is part of the core microbiome.

**Figure 1.** Bathymetric map of the study area off Anvers Island. *Synoicum adareanum* collection sites are shown with red triangles. The map was generated by Environmental Research and Assessment, Cambridge, UK, using Arthur Harbor bathymetry data from the PRIMO survey project 2004–2006 (Dr. Scott Gallagher and Dr. Vernon Asper). Inset: Colonial ascidian, *S. adareanum*, which occurs in clusters of multiple lobes connected by a peduncle which together comprise a colony on the seafloor, collected at depths ranging from 24 to 31 m.
