Horizontal gene transfer in asgard archaea

Horizontal Gene Transfer in Asgard Archaea Saioa Manzano-Morales∗†, Toni Gabald´ on∗†‡ ∗Barcelona Supercomputing Center, Barcelona, Spain †Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain ‡Instituci´ on Catalana de Investigaci´ on y Estudios Avanzados, Barcelona, Spain E-mail: [email protected], [email protected] Keywords—Horizontal Gene Transfer, Asgard Archaea, Reticulate evolution, eukaryogenesis. I. EXTENDED ABSTRACT Asgard archaea are considered to be the closest prokaryotic relative of eukaryotes [1]. They harbor many of what were previously thought to be eukaryote-exclusive proteins [1], including actin and actin-related proteins [2], and the presence of an actin cytoskeleton in particular has been proven in an isolated Lokiarchaeum [3]. As such, they are a key player in the debate surrounding the origin of eukaryotes (a process called eukaryogenesis) [4]. Being prokaryotes, the genome evolution of the Asgard Archaea is likely to have been shaped in no small part by Horizontal Gene Transfer (HGT), that is, the transfer of genetic material between organisms that are not bound by a parentoffspring relationship [5]. These transferred genes often encode for proteins that are beneficial for the cell and allow for adaptation to new niches [6]. In this work, we aim to unveil the fraction of the Asgard protein repertoire that stems from horizontal transfer events, by applying a HGT detection pipeline that combines homologybased and phylogeny-based methods. By analyzing the functional categories and putative donors of these genes, we hope to understand more about the evolution of Asgard archaeal genomes, so that we can employ this knowledge to shed light on the putative ecology and relationships of the archaeal partner of the symbiosis that would give rise to eukaryotic cells. A. HGT detection pipeline The genomic sequences and protein predictions for the cultured isolates Candidatus Prometheoarchaeum syntrophicum MK-D1 [7] (assembly accession GCF-008000775.1) and Candidatus Lokiarchaeum ossiferum/Lokiarchaeum sp. B-35 [3] (GenBank code CP104013.1) were downloaded from NCBI Assembly and NCBI Nucleotide/Protein, respectively. We performed a similarity search with BLAST 2.11.0 [8] of the proteomes against a custom-made database comprised of all the species representatives of the Genome Taxonomy Database [9] species representatives and proteomes from a curated set of eukaryotes, to obtain a sufficiently representative sampling of protein sequences across the Tree of Life. We parsed the BLAST results with HGTector [10], which systematically analyzes BLAST results looking for hit distribution patterns incongruent with a vertical evolution, given a series of hierarchically defined evolutionary categories. This step Fig. 1. HGT detection pipeline identified putative horizontally-transferred genes: for those, we retrieved the best 150 hits and reconstructed a gene tree following the algorithm implemented for PhylomeDB [11]. We further analyzed the resulting gene trees with Abaccus [12], which identifies taxonomical “jumps” in gene trees that do not follow the species tree and therefore further helps discern putative HGT events. Lastly, we performed a manual curation with an ete3-based in-house script [13] to further filter out false positives and to assess the acceptor and donor clades. B. Results Table I displays the number of putatively transferred genes per step in the pipeline and organism. 9.39% and 6.94% of the protein content of Ca. Lokiarchaeum ossiferum and Ca. Prometheoarchaeum syntrophicum, respectively, is of bacterial origin. The transfer events have occurred over a series of timepoints across the Asgard lineage 2: from genus-level to transfers that precede the diversification of the Loki lineage. Interestingly, there is a high degree of paraphyly, with many instances of the Asgard lineage forming two (or more) clades: one that branches close to Archaea (therefore, likely a copy of vertical inheritance) and one that branches closer to a bacterial clade (therefore, a likely transfer). This implies some degree TABLE I. NUMBER OF HORIZONTALLY TRANSFERRED GENES) Organism Prot. HGTector Abaccus HGTs Ca. L. ossiferum 5119 717 513 481 (442) Ca. P. syntrophicum 3890 432 359 270 (256) ossiferum syntrophicum Lokiarchaeum AMARA-1 CR-4 Lokiarchaeia Asgardarchaeota Prometheoarchaeum AMARA-1 CR-4 Lokiarchaeia Asgardarchaeota 0 30 60 90 120 Transfer partner Number of HGT trees monophyly_lineage False True A ossiferum syntrophicum Lokiarchaeum AMARA-1 CR-4 Lokiarchaeia Asgardarchaeota Prometheoarchaeum AMARA-1 CR-4 Lokiarchaeia Asgardarchaeota 0 30 60 90 120 Transfer partner Number of HGT trees monophyly_Asgard False True B Fig. 2. Barplot displaying the number of trees per transfer acceptor. (A) Monophyly of the acceptor lineage. (B) Monophyly of the Asgard archaea. of substitution of vertically-inherited copies by transferred ones, and a co-existence of both sources across the Asgard clade. Independent transfer events also cannot be ruled out. These transfers come from a wide arrange of donor phyla, with prominent donors being Firmicutes and Chloroflexota, followed by Proteobacteria, Spirochaeota, Desulfobacteriota and Bacteroidota. The contribution of Desulfobacterota is particularly interesting, as sulfate-reducing bacteria are known syntrophic partners of these Asgard archaea. The contribution of Anaerolineae within Chloroflexota is also non-trivial, since this lineage is known to inhabit marine sediments, a habitat where these Lokiarchaeia have been sampled. We found instances of both Bacteria-to-Asgard and Asgardto-Bacteria transfer, implying bidirectional flow between transfer partners. Transferred genes seem to be enriched in metabolic functions, mainly related to lipid and amino acid metabolism, functions that seem central to the functions of the cell. They seem to mainly be components of the membrane (GO:0016021), and there is a high degree of overlap between both Lokiarchaeia. C. Conclusion In this study, we observe HGT events to be widespread across Asgard evolution, constituting a continuous flow of transferred genes at different points in the diversification of these archaea, and coming from a variety of donors, some of which can be linked by a metabolic or ecologic relationship. II. ACKNOWLEDGMENT This research was supported by Gordon and Betty Moore Foundation (Grant GBMF9742). REFERENCES [1] K. Zaremba-Niedzwiedzka et al., “Asgard archaea illuminate the origin of eukaryotic cellular complexity,” Nature, vol. 541, no. 7637, pp. 353– 358, Jan. 2017. [2] C. Akıl and R. C. Robinson, “Genomes of asgard archaea encode profilins that regulate actin,” Nature, vol. 562, no. 7727, pp. 439–443, Oct. 2018. [3] T. Rodrigues-Oliveira et al., “Actin cytoskeleton and complex cell architecture in an asgard archaeon,” Nature, vol. 613, no. 7943, pp. 332–339, Jan. 2023. [4] E. V. 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Zhu et al., “HGTector: an automated method facilitating genomewide discovery of putative horizontal gene transfers,” BMC Genomics, vol. 15, p. 717, Aug. 2014. [11] D. Fuentes et al., “PhylomeDB v5: an expanding repository for genomewide catalogues of annotated gene phylogenies,” Nucleic Acids Res., vol. 50, no. D1, pp. D1062–D1068, Jan. 2022. [12] M. A. Naranjo-Ort´ ız et al., “Widespread interand intra-domain horizontal gene transfer of d-amino acid metabolism enzymes in eukaryotes,” Front. Microbiol., vol. 7, p. 2001, Dec. 2016. [13] J. Huerta-Cepas et al., “ETE 3: Reconstruction, analysis, and visualization of phylogenomic data,” Mol. Biol. Evol., vol. 33, no. 6, pp. 1635–1638, Jun. 2016. Saioa Manzano-Morales received his BSc degree in Biochemistry and Molecular Biology from the University of the Basque Country (UPV-EHU), Spain in 2019. She then completed her MSc degree in Computational Biology from the Politechnical University of Madrid, Spain in 2021. After a brief internship in the CIB Margarita Salas (CSIC), she has been with the Comparative Genomics group of Barcelona Supercomputing Center (BSC), where she is developing her PhD.