title: A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae).
abstract: Classical Sanger sequencing is still frequently used to generate sequence data for phylogenetic and phylogeographic inference. In this contribution we present a novel approach to genotype whole mitogenomic haplotypes using Illumina MiSeq reads from indexed amplicons. Our new approach reduces preparation time by multiplexing loci within a single or few PCR reactions and by plate format library construction. The use of paired-end reads allows covering amplicons of about 0.5kb and thus no nebulisation and assembly are necessary. We tested the power and effectiveness of this technique by analysing the mitogenomic diversity of East African white-eye bird species (Zosteropidae), a taxonomically highly diverse and complex species flock found in various ecosystems spread across major parts of Africa. We compare the newly generated mitogenomic data set with published data of three mitochondrial genes for a similar set of populations and taxa. The comparison demonstrates that our new procedure represents a cost effective use of NGS for medium throughput phylogenetic analyses. Using this method, we were able to increase the amount of phylogenetic information significantly, while reducing the costs and effort in the laboratory. The mitogenomic data show a higher resolution than previous studies providing higher support and new insights in the relationships of Zosterops species. Our data suggest to split Z. poliogaster into four distinct species, three of which had previously been proposed: Z. silvanus, Z. mbulensis, Z. kikyuensis and Z. kulalensis. Our approach allows the genotyping of whole mitogenomes for a large number of individuals and thus allows more reliable reconstruction of phylogenetic and phylogeographic relationships - also for non-model organisms.
authors.affiliation: Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences (Boku), A-1180 Vienna, Austria. Electronic address: meimberg@boku.ac.at.
authors.forename: Harald
authors.initials: H
authors.lastname: Meimberg
authors.affiliation: Chair of Restoration Ecology, Department of Ecology and Ecosystem Management, School of Life Sciences Weihenstephan, Technische Universität München, D-85354 Freising, Germany; Cell Biology, Department of Biology, University of Erlangen-Nuremberg, D-91058 Erlangen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Schachtler
authors.affiliation: Research Center in Biodiversity and Genetic Resources (CIBIO)/InBio Associated Laboratory, University of Porto, Campus Vairão, R. Monte-Crasto, P-4485-661 Vairão, Portugal.
authors.forename: Manuel
authors.initials: M
authors.lastname: Curto
authors.affiliation: General Zoology, Institute of Biology, Martin-Luther University Halle-Wittenberg, D-06120 Halle, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Husemann
authors.affiliation: Terrestrial Ecology Research Group, Department of Ecology and Ecosystem Management, School of Life Sciences Weihenstephan, Technical University Munich, D-85354 Freising, Germany.
authors.forename: Jan Christian
authors.initials: JC
authors.lastname: Habel
mesh_terms: D000349:Africa; D000818:Animals; D001483:Base Sequence; D001499:Bayes Theorem; D054629:Genome, Mitochondrial; D046109:Passeriformes / Q000145:classification* / Q000235:genetics*; D010802:Phylogeny; D058974:Phylogeography; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
keywords: Amplicon sequencing; Illumina; MiSeq; Mitogenomics; Radiation; Zosterops