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This approximation of best matches by best hits becomes exact for ultrametric dissimilarities, i.e., under the Molecular Clock Hypothesis. It fails, however, whenever there are large lineage specific rate variations among paralogous genes. In practice, this introduces a high level of noise into the input data for best-hit-based orthology detection methods.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>If additive distances between genes are known, then evolutionary most closely related pairs can be identified by considering certain quartets of genes provided that in each quartet the outgroup relative to the remaining three genes is known.<jats:italic>A priori<\/jats:italic>knowledge of underlying species phylogeny greatly facilitates the identification of the required outgroup. Although the workflow remains a heuristic since the correct outgroup cannot be determined reliably in all cases, simulations with lineage specific biases and rate asymmetries show that nearly perfect results can be achieved. In a realistic setting, where distances data have to be estimated from sequence data and hence are noisy, it is still possible to obtain highly accurate sets of best matches.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Improvements of tree-free orthology assessment methods can be expected from a combination of the accurate inference of best matches reported here and recent mathematical advances in the understanding of (reciprocal) best match graphs and orthology relations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Availability<\/jats:title><jats:p>Accompanying software is available at<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/david-schaller\/AsymmeTree\">https:\/\/github.com\/david-schaller\/AsymmeTree<\/jats:ext-link>.<\/jats:p><\/jats:sec>","DOI":"10.1186\/s13015-020-00165-2","type":"journal-article","created":{"date-parts":[[2020,4,9]],"date-time":"2020-04-09T14:02:57Z","timestamp":1586440977000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["From pairs of most similar sequences to phylogenetic best matches"],"prefix":"10.1186","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5016-5191","authenticated-orcid":false,"given":"Peter F.","family":"Stadler","sequence":"first","affiliation":[]},{"given":"Manuela","family":"Gei\u00df","sequence":"additional","affiliation":[]},{"given":"David","family":"Schaller","sequence":"additional","affiliation":[]},{"given":"Alitzel","family":"L\u00f3pez S\u00e1nchez","sequence":"additional","affiliation":[]},{"given":"Marcos","family":"Gonz\u00e1lez Laffitte","sequence":"additional","affiliation":[]},{"given":"Dulce I.","family":"Valdivia","sequence":"additional","affiliation":[]},{"given":"Marc","family":"Hellmuth","sequence":"additional","affiliation":[]},{"given":"Maribel","family":"Hern\u00e1ndez Rosales","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2020,4,9]]},"reference":[{"key":"165_CR1","doi-asserted-by":"publisher","first-page":"99","DOI":"10.2307\/2412448","volume":"19","author":"WM Fitch","year":"1970","unstructured":"Fitch WM. 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