Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

Department of Biogeography Faculty of Spatial and Environmental Sciences University of Trier Universitätsring 15 Trier 54296 Germany

Department of Biogeography Faculty of Spatial and Environmental Sciences University of Trier Universitätsring 15 Trier 54296 Germany; Department of Botany and Zoology Faculty of Science Masaryk University Kotlářská 2 Brno 611 37 Czech Republic

Ecology and Genetics Research Unit University of Oulu Pentti Kaiteran katu 1 Linnanmaa Finland

Evolutionary Biomechanics Zoological Institute and Museum University of Greifswald Loitzer Str 26 Greifswald 17489 Germany; School of Natural Sciences Macquarie University NSW 2109 Sydney Australia

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