The impact of fossil calibrations, codon positions and relaxed clocks on the divergence time estimates of the native Australian rodents (Conilurini) Maria A. Nilsson a,b, ⁎, Anna Härlid b , Morgan Kullberg b , Axel Janke c a Institute of Experimental Pathology (ZMBE), University of Münster, Von-Esmarch-Str. 56, D-48149 Münster, Germany b Department of Cell and Organism Biology, Division of Evolutionary Molecular Systematics, University of Lund, Sölvegatan 35, S-223 62 Lund, Sweden c Biodiversität und Klima Forschungszentrum BiK-F, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany abstract article info Article history: Received 16 October 2009 Received in revised form 4 February 2010 Accepted 4 February 2010 Available online 11 February 2010 Received by M. Batzer Keywords: Rodentia Leggadina Pseudomys Australia Molecular divergence times Mus–Rattus calibration point The native rodents are the most species-rich placental mammal group on the Australian continent. Fossils of native Australian rodents belonging to the group Conilurini are known from Northern Australia at 4.5 Ma. These fossil assemblages already display a rich diversity of rodents, but the exact timing of their arrival on the Australian continent is not yet established. The complete mitochondrial genomes of two native Australian rodents, Leggadina lakedownensis (Lakeland Downs mouse) and Pseudomys chapmani (Western Pebble-mound mouse) were sequenced for investigating their evolutionary history. The molecular data were used for studying the phylogenetic position and divergence times of the Australian rodents, using 12 calibration points and various methods. Phylogenetic analyses place the native Australian rodents as the sister-group to the genus Mus. The Mus– Conilurini calibration point (7.3–11.0 Ma) is highly critical for estimating rodent divergence times, while the influence of the different algorithms on estimating divergence times is negligible. The influence of the data type was investigated, indicating that amino acid data are more likely to reflect the correct divergence times than nucleotide sequences. The study on the problems related to estimating divergence times in fast-evolving lineages such as rodents, emphasize the choice of data and calibration points as being critical. Furthermore, it is essential to include accurate calibration points for fast-evolving groups, because the divergence times can otherwise be estimated to be significantly older. The divergence times of the Australian rodents are highly congruent and are estimated to 6.5–7.2 Ma, a date that is compatible with their fossil record. © 2010 Elsevier B.V. All rights reserved. 1. Introduction A unique mammalian fauna evolved in Australia due to approx- imately 50 million years (Myr) of isolation. Australia is home to the marsupials and monotremes, which have for various reasons gone extinct on most other continents, except South America. In contrast, placental mammals apart from bats did not reach Australia until recently, allowing the successful radiation of marsupial mammals. Two placental mammalian groups have been thriving in Australia, namely the bats and the murine rodents. Today Australia is home to over 70 species of native rodents (Musser and Carleton, 1993; Strahan, 1995; Tate, 1951; Watts and Aslin, 1981). The Australian rodents are traditionally divided into three groups: Conilurini, Uromyini and Hydromyini (Musser and Carleton, 1993; Watts and Aslin, 1981; Baverstock, 1984). The Conilurini (the pseudo-mouse group) is the most species-rich group and has a strictly Australian distribution. The genera Uromyini and Hydromyini have their main distribution in New Guinea, but some species of these genera – Uromys, Melomys, Hydromys, Xeromys – are also found on the Australian continent. The fossil record of the native Australian rodents, the Conilurini, starts at 4.5 Ma (million years ago), but how and when the earliest rodents reached the Australian continent remains controversial. Australia has been an isolated continent since the last connection with Antarctica was severed in the Eocene. While Australia was drifting northwards and further away from Antarctica, it was getting closer to the Indonesian archipelago. At 5–10 Ma Australia and New Guinea came close enough to the Indonesian islands to facilitate the dispersal of animals (Hall, 2002). It is most likely that at this time the ancestral rodents migrated from South East Asia via Indonesia to the Australian continent. An older analysis of the fossil record suggests that the native rodents reached Australia in three waves (Baverstock, 1984; Hand, 1984). The first wave occurred 4.5 Ma and brought the Conilurini to Australia. Several 4.5 Myr old Pliocene localities such as Gene 455 (2010) 22–31 Abbreviations: Myr, million years; Ma, million years ago; mt, mitochondrial; kb, kilobase; nt, nucleotide; ca, amino acid; ML, maximum likelihood; GTR, general time reversible; MTD, Multidivtime; S. D., standard deviation; cdp, codon position. ⁎ Corresponding author. Institute of Experimental Pathology (ZMBE), University of Münster, Von-Esmarch-Str. 56, D-48149 Münster, Germany. Tel.: + 49 251 8352132; fax: +49 251 8352134. E-mail address: mnilsson@uni-muenster.de (M.A. Nilsson). 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.02.002 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene