A molecular diagnostic for identifying central African forest artiodactyls from faecal pellets
S. Ntie
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorA. R. Johnston
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorP. Mickala
Departement de Biologie, Université des Sciences et Techniques de Masuku, Franceville, Gabon
Search for more papers by this authorA. E. Bowkett
Whitley Wildlife Conservation Trust, Paignton Zoo, Paignton, UK
School of Biosciences, University of Exeter, Exeter, UK
Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, Matieland, South Africa
Search for more papers by this authorB. Jansen van Vuuren
Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, Matieland, South Africa
Search for more papers by this authorM. Colyn
Laboratory of Ethology, Evolution and Ecology, University of Rennes I, CNRS, UMR, Station Biologique de Paimpont, France
Search for more papers by this authorP. Telfer
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
Centre International de Recherches Médicales de Franceville, Franceville, Gabon
Search for more papers by this authorF. Maisels
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
School of Biological and Environmental Sciences, University of Stirling, Stirling, Scotland, UK
Search for more papers by this authorO. Hymas
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
Search for more papers by this authorR. L. Rouyer
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorR. A. Wallace
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorK. LeBlanc
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorN. Van Vliet
Center for International Forestry Research, Yaoundé, Cameroon
Search for more papers by this authorG. Sonet
Royal Belgian Institute of Natural History, Brussels, Belgium
Search for more papers by this authorE. Verheyen
Royal Belgian Institute of Natural History, Brussels, Belgium
Search for more papers by this authorD. Pires
Life Sciences Core Curriculum, University of California, Los Angeles, CA, USA
Search for more papers by this authorE. J. Wickings
Centre International de Recherches Médicales de Franceville, Franceville, Gabon
Search for more papers by this authorS. A. Lahm
lnstitut de Recherche en Ecologie Tropicale, Makokou, Gabon
Ecology and Environment Inc., Lancaster, NY, USA
Search for more papers by this authorN. M. Anthony
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorS. Ntie
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorA. R. Johnston
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorP. Mickala
Departement de Biologie, Université des Sciences et Techniques de Masuku, Franceville, Gabon
Search for more papers by this authorA. E. Bowkett
Whitley Wildlife Conservation Trust, Paignton Zoo, Paignton, UK
School of Biosciences, University of Exeter, Exeter, UK
Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, Matieland, South Africa
Search for more papers by this authorB. Jansen van Vuuren
Department of Botany and Zoology, University of Stellenbosch, Stellenbosch, Matieland, South Africa
Search for more papers by this authorM. Colyn
Laboratory of Ethology, Evolution and Ecology, University of Rennes I, CNRS, UMR, Station Biologique de Paimpont, France
Search for more papers by this authorP. Telfer
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
Centre International de Recherches Médicales de Franceville, Franceville, Gabon
Search for more papers by this authorF. Maisels
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
School of Biological and Environmental Sciences, University of Stirling, Stirling, Scotland, UK
Search for more papers by this authorO. Hymas
Wildlife Conservation Society, 2300 Southern Boulevard, New York, NY, USA
Search for more papers by this authorR. L. Rouyer
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorR. A. Wallace
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorK. LeBlanc
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorN. Van Vliet
Center for International Forestry Research, Yaoundé, Cameroon
Search for more papers by this authorG. Sonet
Royal Belgian Institute of Natural History, Brussels, Belgium
Search for more papers by this authorE. Verheyen
Royal Belgian Institute of Natural History, Brussels, Belgium
Search for more papers by this authorD. Pires
Life Sciences Core Curriculum, University of California, Los Angeles, CA, USA
Search for more papers by this authorE. J. Wickings
Centre International de Recherches Médicales de Franceville, Franceville, Gabon
Search for more papers by this authorS. A. Lahm
lnstitut de Recherche en Ecologie Tropicale, Makokou, Gabon
Ecology and Environment Inc., Lancaster, NY, USA
Search for more papers by this authorN. M. Anthony
Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA
Search for more papers by this authorNicola Anthony, Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA.
Email: [email protected]
Abstract
Small to medium-sized central African forest artiodactyls constitute a diverse yet heavily hunted group composed primarily of species within the genera Cephalophus, Neotragus, Tragelaphus and Hyemoschus. Of these genera, Cephalophus is the richest with as many as seven sympatric species known to occur in central African forests. However, differentiating species from their faeces or from tissue where the whole carcass is unavailable is very difficult. In order to develop a robust molecular diagnostic for species identification, a database of mitochondrial cytochrome b (553 bp) and control region (∼675 bp) sequences was compiled from all forest Cephalophus species and other similarly sized, sympatric Tragelaphus, Neotragus and Hyemoschus species. Reference phylogenies from each marker were then used to recover the identity of sequences obtained from unknown faecal samples collected in the field. Results were then compared to determine which region best recovered species identity with the highest statistical support. Restriction fragment length polymorphisms (RFLPs) were also assessed as an alternative method for rapid species identification. Of the methods examined, tree-based analyses built on a geographically comprehensive database of control region sequences was the best means of reliably recovering species identity from central African duikers. However, three sister taxa appear indistinguishable (Cephalophus callipygus, Cephalophus ogilbyi and Cephalophus weynsi) and not all species were monophyletic. This lack of monophyly may be due to incomplete lineage sorting commonly observed in recently derived taxa, hybridization or the presence of nuclear translocated copies of mitochondrial DNA. The high level of intra-specific variation and lack of robust species-specific diagnostic sites made an RFLP-based approach to duiker species identification difficult to implement. The tree-based control region diagnostic presented here has many important applications including fine-scale mapping of species distributions, identification of confiscated tissue and environmental impact assessments.
Supporting Information
Figure S1. Cytochrome b neighbour-joining bootstrap consensus phylogeny based on Kimura-2-parameter corrected distances and rooted with H. aquaticus. Sequences were aligned with CLUSTAL. Bootstrap values are indicated at the relevant node.
Figure S2. Cytochrome b maximum parsimony bootstrap consensus phylogeny rooted with H. aquaticus. Sequences were aligned with CLUSTAL. Bootstrap values are indicated at the appropriate node.
Figure S3. Control region phylogeny based on Bayesian analysis and rooted with Tragelaphus species. Sequences were aligned with CLUSTAL. Posterior probability values are indicated at the appropriate node.
Figure S4. Control region neighbour-joining bootstrap consensus phylogeny based on Kimura-2-parameter corrected distances and rooted with N. moschatus. Sequences were aligned with CLUSTAL. Bootstrap values are indicated at the relevant node.
Figure S5. Control region maximum parsimony bootstrap consensus phylogeny rooted with N. moschatus. Sequences were aligned with CLUSTAL. Bootstrap values are indicated at the appropriate node.
Figure S6. Flow chart illustrating the application of RFLP banding patterns to the diagnosis of species using cytochrome b gene sequence data.
Table S1. Details of the species, provider, geographic origin and GenBank accession numbers for samples used in the present study.
Table S2. Candidate cytochrome b gene RFLPs for diagnosing central African rainforest ungulate species and the size fragments (indicated in parentheses) generated by each enzyme.
Table S3. Candidate control region RFLPs for diagnosing central African rainforest ungulate species and the size fragments (indicated in parentheses) generated by each.
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