Anonymous nuclear markers for the African adders (Serpentes: Viperidae: Bitis )
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TECHNICAL NOTE
Anonymous nuclear markers for the African adders (Serpentes: Viperidae: Bitis) Axel Barlow • Wendy Grail • Mark de Bruyn Wolfgang Wu¨ster
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Received: 16 May 2012 / Accepted: 30 May 2012 / Published online: 12 June 2012 Ó Springer Science+Business Media B.V. 2012
Abstract We report five novel anonymous nuclear loci for the African viper genus Bitis, developed from a genomic library of a single individual puff adder (Bitis arietans). All loci amplify consistently and are variable among seven B. arietans sampled from disparate portions of the geographic range. Trials with nine congeneric species showed one locus amplifies across all species tested, three amplify in a subset of species and one only amplifies in B. arietans. Genetic divergences for these loci range from comparable, to more than double that of a commonly used nuclear protein-coding marker in Bitis. These loci will provide a valuable resource for future investigations of these snakes. Keywords Anonymous nuclear loci Phylogenetics Phylogeography Africa Bitis
The use of multiple independent loci is now standard practise for phylogenetic and phylogeographic studies (Heled and Drummond 2010), however investigations at lower taxonomic levels may be hampered by a lack of availability of sufficiently variable nuclear markers (Brito and Edwards 2009). We report the development of five single-copy non-coding anonymous nuclear markers for the African viper genus Bitis to facilitate such studies. The genus Bitis, commonly referred to as the African adders, represents Africa’s most taxonomically diverse and geographically widespread genus of viperid snakes (Branch
A. Barlow (&) W. Grail M. de Bruyn W. Wu¨ster School of Biological Sciences, Environment Centre Wales, Bangor University, Bangor LL57 2UW, UK e-mail: [email protected]
1998), and includes well known and iconic large-bodied species such as the gaboon vipers (B. gabonica, B. rhinoceros) and medically significant puff adder (B. arietans). The genus also contains numerous smaller representatives, many of which have extremely restricted distributions within which several are threatened by habitat destruction and illegal collection for the pet trade (Branch 1999). Furthermore, two species (B. inornata, B. schneideri) are classified as vulnerable in the IUCN red list of threatened species (IUCN 2011). We assembled a genomic library from a single B. arietans individual from Hartebeespoort, South Africa. Approximately 12 lg of genomic DNA was extracted from a large scale clipping using the high-salt method (Aljanabi and Martinez 1997) and randomly fragmented using the enzyme Rsa1. The resulting digest was visualised on a 1 % agarose gel and fragments within the range 0.5–1.5 kb excised and purified by gel extraction (Qiagen cat. no. 28704, eluted in 50 lL HPLC grade H20). Size selected DNA was then centrifuged to achieve a final concentration [20 ng/lL and approximately 100 ng ligated into 25 ng of PCR Zero Blunt vector and transformed into competent One Shot TOP10 Escherichia c
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