experiences low pathogen pressure, accounting for their disease susceptibility in foreign 28 environments. However, there is a limited understanding of virome diversity in Antarctic 29 species, the extent of in situ virus evolution, or how it relates to that in other geographic 30 regions. To test the idea that penguins have limited microbial diversity we determined the 31 viromes of three species of penguins and their ticks sampled on the Antarctic peninsula. 32 Using total RNA-Sequencing we identified 107 viral species, comprising likely penguin 33 associated viruses (n = 13), penguin diet and microbiome associated viruses (n = 82) and tick 34 viruses (n = 8), two of which may have the potential to infect penguins. Notably, the level of 35 virome diversity revealed in penguins is comparable to that seen in Australian waterbirds, 36 including many of the same viral families. These data therefore reject the theory that 37 penguins are subject to lower pathogen pressure. The repeated detection of specific viruses in 38 Introduction 43 Geographical separation and extreme climates have resulted in the long isolation of 44 Antarctica and the subantarctic islands. The result is a unique assemblage of animals, some 45 relying entirely on the frozen continent, with others utilizing the fringes. Such geographic 46 isolation has been proposed to explain why Antarctic fauna supposedly harbour a paucity of 47 viruses, and supported by the observation that captive Antarctic penguins are highly 48 susceptible to infectious diseases (1). It has therefore been hypothesized that Antarctic fauna 49 have evolved in a setting of low "pathogen pressure", reflected in limited microbial diversity 50 and abundance (1, 2). As a consequence, the potential for climate driven and human mediated 51 movement of microorganisms makes the expansion of infectious diseases to the Antarctic a 52 matter of considerable concern (1, 3-5). 53 To date, a small number of viral species have been described in Antarctic fauna (6). 54 Serological studies have revealed that Antarctic penguins are reservoirs for influenza A virus 55 (IAV), avian avulaviruses (formerly avian paramyxoviruses), birnaviruses, herpesviruses, and 56 flaviviruses (7-13). Despite improvements in the molecular tools for virus detection, it is only 57 in recent years that full viral genomes have been characterized (6). For example, 58 adenoviruses, astroviruses, paramyxoviruses, orthomyxoviruses, polyomaviruses and 59 papillomavirus have been identified in Adélie penguins (Pygoscelis adeliae), Chinstrap 60 penguins (Pygoscelis antarctica) and Gentoo penguins (Pygoscelis papua) (6, 14-22). 61 However, sampling is limited and genomic data sparse, such that we have a fragmented 62 understanding of virus diversity in penguins and in Antarctica in general. 63 Antarctic penguins may also be infected by viruses spread by ectoparasites, particularly the 64 seabird tick Ixodes uriae (White) (23). For example, seven different arthropod-borne viruses 65 (arboviruses) were identified in I. uriae ticks collected from King nymphs) were collected from Paradise Bay (Fig 1) . Ticks were collected under rocks within 106 and directly adjacent to penguin colonies and placed in RNAlater (Ambion) and stored at -107 80°C within 4-8 hours of collection. 108 RNA library construction and sequencing 109 RNA library construction, sequencing and RNA virus discovery was carried out as described (Table S1 ). Sequence reads were demultiplexed and trimmed with Trimmomatic followed by de novo 131 assembly using Trinity (34). No filtering of host/bacterial reads was performed before 132 assembly. All assembled contigs were compared to the entire non-redundant nucleotide (nt) 133 and protein (nr) database using blastn and diamond blast (35), respectively, setting an e-value 134 threshold of 1x10 -10 to remove false-positives. Abundance estimates for all contigs were determined using the RSEM algorithm (34). All 136 contigs that returned blast hits with paired abundance estimates were filtered to remove plant, 137 invertebrate fungal, bacterial and host sequences. Viruses detected in the penguin libraries 138 were divided into those likely to infect birds and those likely associated other hosts (36)(37). 139 This division was performed using a combination of phylogenetic analysis and information (Table S1 ). There was a large range in the total viral 185 abundance in both the penguin (0.07-0.7 % total viral reads; 0-0.15 % avian viral reads) and 186 tick libraries (0.03-2.4%) (Table S1, Fig 2) . In addition to likely avian viruses, the penguin 187 libraries contained numerous reads matching insect, plant, or bacterial viruses and 188 retroviruses (Fig 2A, Fig 3) . Retroviruses were excluded from later analyses due to the 189 challenges associated with differentiating exogenous from endogenous sequences using meta- sampling locations that are 130 km apart (Fig 3, Fig S3) . 213 Within the tick libraries, the greatest virus abundance was seen in the adult female ticks, Paramyxoviridae, Picorbirnaviridae, Picornaviridae and Reoviridae (Fig 2B, Fig 3) (see 229 below). Ten of the 13 avian associated viruses identified in the penguins likely represent 230 9 novel avian viral species (Table S2, Fig 4) Table S3 , Fig 3) . The largest diversity was found in the "Narna-Levi", "Noda-Tombus" and 245 "Picorbirna-Pariti" viral groups (36). A number of viruses were highly divergent, including 246 clusters of novel viruses that fell within the Narnaviridae and Leviviridae (Table S3, Fig 3) . (Fig 6A, Fig S9) We also identified a deltacoronavirus and an avastrovirus (Fig 6B, Fig S10-S11 ). The 294 deltacoronavirus was similar to those reported in birds in the United Arab Emirates, 295 Australia, Niger, and Finland, with ~95% identity. A lack of sampling makes it challenging to 296 determine how deltacoronaviruses in Antarctica and other continents may be shared (Fig   297   S10 ). The astrovirus detected was similar (88.3% identity) to a short fragment (1000 bp) 298 previously reported in Adelie penguins on the Ross ice shelf of Antarctica (22) (Table S2) , a 299 pattern confirmed by phylogenetic analysis (Fig 6B) . Phylogenetic analysis also reveals that 300 this virus falls in an outgroup to Group 2 viruses, including Avian Nephritis virus (Fig 6B, 301 Fig S11) . Although we were unable to determine the epidemiology of these viruses in 302 Antarctica, repeated detection on opposite ends of the Antarctic continent makes it possible 303 that this is a penguin specific virus. (Fig 4) . 320 The six other novel virus species identified in the tick libraries comprised five viral families: RdRp sequences currently available, exhibiting just 35.7% amino acid sequence similarity to 328 the divergent tick-borne tetravirus-like virus (Fig S12) . A novel colti-like virus (Reoviridae), 329 Fennes virus, was identified in the adult male, female and nymph libraries, although we were 330 only able to assemble four segments. Notably, Fennes virus falls basal to the existing 331 coltivirus group, exhibiting just 30% amino acid sequence similarity to Shelly headland virus, 332 recently identified in Ixodes holocyclus ticks from Australia (Fig 7) . The partial genome of a 333 Rhabdovirus, Messner virus, was identified in the adult female library. However, this 334 fragment was of low abundance, and only the RdRp segment (Fig S12) . (Table S2) . Combined, these data strongly suggest that penguins are not merely spill-over hosts, but may 379 be central reservoir hosts for a diverse range of viruses. This is apparent in two observations. 380 The first is the repeated detection of specific virus species, such as avian avulaviruses, and In sum, we reveal substantial viral diversity in Antarctic penguins, their diet and their ticks. 433 We therefore expect that additional viruses will be identified with increased sampling, Table S3 , and phylogenetic 632 trees for each virus family can be found in Figs 5-8, S5-S12). 633 