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Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904–3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica. [ABSTRACT FROM AUTHOR]

Highlights: [•] Identification of 13 novel ssDNA viruses in P. grayi and X. zealandica. [•] The 13 novel viral genomes (1628–2668nt) have NAGTATTAC nonanucleotide motif. [•] The novel ssDNA viruses have 2 ORFs that are bidirectional or unidirectional. [•] Reps of novel ssDNA viruses have conserved the RCR & helicase motifs. [Copyright &y& Elsevier]