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Metagenome-Assembled Genomes (MAGs) have been important objects of study in the understanding of entire microbial communities in specific environments. According to Setubal (2021), MAGs can be classified in two ways: Species MAG (SMAG) – a MAG classified at the species level and for which there is at least one genome of isolate as a reference – or Hypothetical MAG (HMAG) – a MAG classified at less specific taxonomic levels and without a genome of an isolate as a reference. Furthermore, for HMAGs, if there is at least one other sufficiently similar genome from a study in a different geographic location, we say that it is a Conserved Hypothetical MAG (CHMAG). Given the growing number of publicly available genomes, as well as the publication of MAG catalogs, in this work we aimed to characterize SMAGs and CHMAGs recovered from three different environments at the São Paulo Zoo (composting [Braga et al. 2021], howler monkey feces [Franco et al., unpublished], and reservoir water [Barbosa et al., unpublished]), comparing them to sufficiently similar genomes, which we call “orthologous genomes”. We classified these MAGs as SMAGs or HMAGs based on results obtained with GTDB-tk (Chaumeil, 2020). Then, using Average Nucleotide Identity (ANI) as a metric, our SMAGs were compared with genomes deposited in GenBank and JGI, and with MAGs from the Genomes from Earth’s Microbiomes (GEM) ( Nayfach, 2021) and Non-Human Primates Gut Microbiome (NHPGM) (Manara, 2019) catalogs. Here we consider orthologous genomes those pairs of genomes for which ANI ≥ 95%. The HMAGs were also compared with the genomes of the catalogs, so we were able to determine that, of the 150 HMAGs in the three environments, 34 are actually CHMAGs. Many orthologous genomes found for MAGs from howler monkey feces are from the human digestive system, while others were recovered from the gut microbiota of both Old World and New World monkeys. For water reservoir MAGs, all orthologous genomes come from freshwater samples, except ZLKRG40 (Burkholderiaceae), which has an ortholog recovered from a saline aquatic environment. Most orthologous genomes of the composting samples were also obtained from composting in other locations or from environments where biomass degradation takes place. We compared orthologous genomes in a set to each other using the MAGset tool, which uses the concept of Genomic Region of Interest (GRI), which is a region of at least 5,000 bp with differential presence between a target genome (our MAG) and one or more reference orthologous genomes. The GRIs existing only in target SMAGs were analyzed to find genes that could indicate adaptations to their environment. Currently, seven SMAGs are being analyzed: ZC3RG05 (Thermobifida fusca), ZC3RG08 (Planifilum fulgidum), ZC4RG04 (Thermobispora bispora), ZC4RG08 (Pseudomonas thermotolerans), ZC4RG13 (Rhodothermus marinus), ZC4RG43 (Mycobacterium hassiacum) and ZC4RG45 (Thermocrispum agreste). All of them are from composting. As an example of this analysis, one GRI gene in ZC4RG04 encodes a GH3 CAZyme family protein that performs a range of functions including cellulosic biomass degradation. Acknowledgments to FAPEMA, which funded part of this work.

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#	Name
1	Robson Oliveira(robsonpontes@ime.usp.br)
2	Fabio Sanchez(fabio.b.sanchez@gmail.com,)
3	Layla Martins(laylabiol@gmail.com)
4	Aline Silva(almsilva@iq.usp.br)
5	João Setubal(setubal@iq.usp.br)

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1	Braga, L., R.V. Pereira, L.F. Martins, L. Moura, F.B. Sanchez, J.S.P. Patané, A.M. da Silva, J.C. Setubal. Genome-Resolved Metagenome And Metatranscriptome Analyses Of Thermophilic Composting Reveal Key Bacterial Players And Their Metabolic Interactions. In BMC Genomics, 22:652, 2021. Chaumeil, P. A., Mussig, A. J., Hugenholtz, P., Parks, D. H. (2020). GTDB-Tk: A Toolkit To Classify Genomes With The Genome Taxonomy Database. In Bioinformatics, pages 1925- 1927. Manara, S., Asnicar, F., Beghini, F., Bazzani, D., Cumbo, F., Zolfo, M., Nigro, E., Karcher, N., Manghi, P., Metzger, M. I., Pasolli, E., & Segata, N. (2019) Microbial Genomes From Non- Human Primate Gut Metagenomes Expand The Primate-Associated Bacterial Tree Of Life With Over 1000 Novel Species. In Genome Biology, pages 1-16. Nayfach, S., Roux, S., Seshadri, R., Udwary, D., Varghese, N., Schulz, F., Wu, D., Paez- Espino, D., Chen, I. M., Huntemann, M., Palaniappan, K., Ladau, J., Mukherjee, S., Reddy, T. B. K., Nielsen, T., Kirton, E., Faria, J. P., Edirisinghe, J. N., Henry, C. S., Eloe-Fadrosh, E. A. (2021). A Genomic Catalog Of Earth’s Microbiomes. In Nature Biotechnology, pages 499-509. Setubal, J.C. (2021). Metagenome-Assembled Genomes: Concepts, Analogies, And Challenges. In Biophysical Reviews, pages 905-909. Publishing Press.

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