Computational protocol: Mitochondrial DNA and karyotypic data confirm the presence of Mus indutus and Mus minutoides (Mammalia, Rodentia, Muridae, Nannomys) in Botswana

Similar protocols

Protocol publication

[…] Our mitochondrial phylogeny was generated from combining previously published sequences deposited on GenBank () with those derived from sequencing new specimens collected during field trips to Botswana conducted in 2008, 2009, and 2011 (, ). We collected 16 specimens of Mus from five localities in Botswana including: Gcwihaba Caves (20°00.99'S, 21°15.89'E); Kang (23°32.10'S, 22°32.76'E); Koanaka Hills (20°09.60'S, 21°11.61'E); Lepokole Hills (21°49.59'S, 28°23.94'E); and Tsabong (25°56.57'S, 22°25.44'E) (). Specimens were collected using Sherman live traps, pitfall traps, or Museum Special snap traps. Standard external measurements were recorded in the field (). Specimens were preserved as skins with complete skeletons (SSPS), skulls only, or as whole bodies in alcohol (alc.) and deposited at the at the Natural Science Research Laboratory (NSRL) at the Museum of Texas Tech University, Lubbock, Texas, USA or the Botswana National Museum, Gaborone, Botswana. Tissue samples were preserved in 95% ethanol, lysis buffer, or flash frozen in liquid nitrogen for future genomic analyses (2011 material) and deposited in the NSRL. Field collecting methods followed taxon specific guidelines for wild mammals () as outlined by the Animal Care and Use Committee of the American Society of Mammalogists (; ).Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen Inc., Chatsworth, California). The complete cytochrome b gene (cytb, 1140 nucleotides) was amplified following methods outlined in . Cycle sequencing reactions were performed with BigDye terminator version 3.1 and were electrophoresed on an ABI 3100-Avant (Applied Biosystems, Foster City, California). Sequences were edited and aligned using SEQUENCHER version 4.9 (Gene Codes Corporation, Ann Arbor, Michigan). Novel sequences (GenBank accession nos. KF184308-KF184323) were aligned with previously published sequences deposited on GenBank using only individuals that exhibited unique haplotypes (). The final alignment was trimmed to exclude regions with large amounts of missing data due to the large number of GenBank sequences in the alignment that were partial cytb sequences. Therefore, a total of 741 base pairs of the cytb gene (the first 7 codons and last 126 codons were removed from the analysis) were used in the final alignment for the phylogenetic analysis including 125 individuals.Appropriate models of evolution were examined using MEGA version 5 (). Phylogenetic relationships were estimated using Bayesian inference with the program MRBAYES version 3.2 (). Four independent Markov chains were run for 50 million generations and trees were logged every 1000th iteration. Log-likelihood values were examined in the program TRACER version 1.5 () and the first 5,000 trees were discarded as burn-in. An additional phylogeny was estimated using the Maximum-likelihood method with the program PhyML version 3.0 () with a BIONJ starting tree () and 1,000 bootstrap replicates. Kimura 2-parameter genetic distances were calculated using MEGA version 5 ().Specimens were karyotyped in the field using bone marrow after 1 h of in vivo incubation with Velban (Sigma-Aldrich, St. Louis, Missouri), following the methods described in . Mus indutus males were not karyotyped in this study because both males captured died in snap traps. Fluorescent in situ hybridization (FISH) experiments were performed using Star*FISH © biotin-labeled mouse chromosome X paints (Cambio), following the manufacturer’s instructions and using Cy3-conjugated streptavidin (Invitrogen) for signal detection.In order to assess the nature of the X-autosome translocation of the specimens that exhibited the translocation, we compared the X-chromosome of our specimens with those from South Africa using images of inverted DAPI-banding, and G-banding (). Images were captured using the GENUS SYSTEM version 3.7 (Applied Imaging Systems, San Jose, California) through an Olympus BX51 epi-fluorescence microscope. Cy3 and DAPI (4’,6-diamidino-2-phenylindole) signals were pseudocolored yellow and red, respectively. […]

Pipeline specifications

Software tools Sequencher, MEGA, MrBayes, PhyML, BIONJ
Application Phylogenetics
Organisms Homo sapiens