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The polyphyly of Neotragus – Results from genetic and morphometric analyses
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Mammalian Biology journal homepage: www.elsevier.com/locate/mambio
Short Communication
The polyphyly of Neotragus – Results from genetic and morphometric analyses Eva V. Bärmann a,b,∗ , Tim Schikora c,d a
Department of Zoology, University of Cambridge, Downing Street, CB2 3EJ, UK Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Invalidenstrasse 43, 10115 Berlin, Germany Biodiversität und Klima Forschungszentrum, 60325 Frankfurt am Main, Germany d Zoo Dortmund, Mergelteichstr. 80, 44225 Dortmund, Germany b c
a r t i c l e
i n f o
Article history: Received 24 September 2013 Accepted 6 January 2014 Handled by Paul Grobler Available online xxx Keywords: Neotragus Cytochrome b Principal component analysis Discriminant analysis Skull morphology
a b s t r a c t Dwarf antelope species were commonly united in the tribe “Neotragini” (Bovidae, Mammalia) due to their general morphological appearance. However, phylogenetic analyses have shown that not all dwarf antelopes are closely related, so it was suggested to restrict the name Neotragini to the type genus Neotragus. In our study we use mitochondrial cytochrome b sequences and linear skull measurements to further investigate the similarity of all three Neotragus species. Our analyses support the close relationship of N. moschatus and N. batesi. However, N. pygmaeus – the type species, which was never before included in phylogenetic analyses – is not closely related. It might share a most recent common ancestor with another “dwarf antelope”, the Klipspringer Oreotragus oreotragus, and the duikers in the taxon Cephalophini. Hence, we suggest resurrecting the genus Nesotragus von Dueben, 1846 for Nesotragus moschatus and N. batesi. © 2014 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
The genus Neotragus C. H. Smith, 1827 comprises the smallest antelopes alive today, with body mass as little as 2 kg in the Royal antelope, Neotragus pygmaeus (Linnaeus, 1758) (Groves and Grubb, 2011). Along with this type species usually two more species are included in Neotragus: the Suni, N. moschatus (von Dueben, 1846), and Bates’ pygmy antelope, N. batesi De Winton, 1903. All three species have an allopatric distribution and inhabit tropical forests of West, Central and East Africa (see Fig. 1A). The Suni was originally placed in its own genus Nesotragus von Dueben, 1846, and later synonymised with Neotragus (Ansell, 1972). Bates’ pygmy antelope on the other hand was usually regarded as congeneric with N. pygmaeus, although Haltenorth (1963) in his “Classification of Mammals” referred it to Nesotragus. Historically, Neotragus was united with other small antelopes like the Klipspringer Oreotragus oreotragus (Zimmermann, 1783), Oribi Ourebia ourebi (Zimmermann, 1783), Beira Dorcatragus megalotis Menges, 1894, the Dikdiks Madoqua spp. Ogilby, 1836 and the Steen- and Grysboks Raphicerus spp. C. H. Smith, 1827 in a group called “Neotragini” (see Nowak 1999). They all have some morphological features in common, like short and straight horns, and show
∗ Corresponding author at: Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Invalidenstrasse 43, 10115 Berlin, Germany. Tel.: +49 0179 2416608. E-mail address: [email protected] (E.V. Bärmann).
a similar social organisation where one male and usually one female share a permanent territory. However, morphological and molecular analyses of the past 20 years have shown that “Neotragini” in the traditional configuration is a polyphyletic assemblage (e.g., Gentry, 1992; Hassanin and Douzery, 1999; Matthee and Davis, 2001; Ropiquet et al., 2009; Hassanin et al., 2012; Schikora, 2012; Bärmann et al., 2013a): Ourebia, Raphicerus, Madoqua, and Dorcatragus are now included in Antilopini, whereas Oreotragus is often placed in its own tribe Oreotragini with yet unresolved phylogenetic relationships. So at present only the genus Neotragus remains in Neotragini. Both N. moschatus and N. batesi are characterised by mitochondrial DNA sequences. In their recent molecular analysis Hassanin et al. (2012) confirmed that they are sister-species, and are presumably closely related to the Impala, Aepyceros melampus (Lichtenstein, 1812). Both these Neotragus species and Aepyceros also share a unique morphological character: a fenestra between premaxillary and maxillary (see Fig. 1B). However, this fenestra is absent in N. pygmaeus. We sequenced the mitochondrial cytochrome b gene of N. pygmaeus in order to investigate its phylogenetic relationship with the other two Neotragus species (in the following called Nesotragus). In addition, we used linear skull measurements in multivariate morphometric analyses to further study the similarity of N. pygmaeus, N. moschatus, N. batesi, O. oreotragus, and four species of Cephalophini.
1616-5047/$ – see front matter © 2014 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.mambio.2014.01.001
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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Fig. 1. (A) Geographic ranges of Neotragus pygmaeus, N. batesi, and N. moschatus in Africa (map re-drawn from IUCN Red List of threatened species www.iucnredlist.org, September 2013); (B) skulls of N. pygmaeus (male, ZSM 1963-143), N. batesi (female, ZSM 1975-28), and N. moschatus (male, ZMB MAM 57144), the fenestra between premaxillary and maxillary in N. batesi and N. moschatus (marked with arrows) is absent in N. pygmaeus; (C) consensus tree from 8000 post burn-in trees of Bayesian analysis of cytochrome b sequences, branch labels = posterior probabilities, Neotragus and Nesotragus are highlighted with bold letters and thick branches, lower case letters correspond to publication references of the sequences: (a) Hassanin et al. (2012), (b) Hassanin et al. (2009), (c) Pitra et al. (1998), (d) Xu et al. (2005), (e) Birungi et al. (1998), and (f) Tungsudjai et al. (no journal reference); (D) bi-variate plot of specimen scores for the two main components in a principal component analysis (PCA) of 23 linear skull measurements; (E) bi-variate plot of specimen scores for two discriminant functions in a discriminant analysis (DA) of 23 linear skull measurements, the discriminant functions derived from the three pre-defined groups Nesotragus, Cephalophini, and Oreotragus are used for classification of N. pygmaeus specimens.
A full blood sample of N. pygmaeus was collected by T.S. from a specimen caught by a local hunter in Ghana (West of Accra, South of Dunkwa Village, along the Kumasi – Yamoransa Rd, GPS coordinates: 5.28838904◦ N, −1.21371042◦ E). It was conserved in 2 vol. ethanol (90%) and stored as cool as possible. DNA was extracted after washing with 500 l CTAB-buffer and overnight incubation
with 20 l proteinase K (10 mg/ml) and 0.65 l -mercaptoethanol at 56 ◦ C, using standard phenol–chloroform extraction (Sambrook and Russell, 2001). The complete cytochrome b sequence (1140 bp) was amplified successfully with the primer pair CytbH and CytbL (Arnason and Gullberg, 1996). The PCR reactions were performed using 0.625 U peqGOLD Taq-DNA-Polymerase (PEQLAB
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s recommendations and performed according to the following protocol: initial denaturation at 94 ◦ C for 3 min, 30 cycles denaturation at 94 ◦ C, annealing at 45 ◦ C, and extension at 72 ◦ C (each step 1 min), and final extension at 72 ◦ C for 10 min. Following the manufacturer’s instructions the PCR products were purified using Sephadex G50 (Sigma–Aldrich, Munich, Germany). The fragments were amplified in forward and in reverse direction in two separate sequencing-reactions using the primers CytbH and CytbL. After ethanol precipitation, sequencing was performed using BigDyeTerminator v.3 on an ABI 3730 DNA Analyzer (Applied Biosystems, Darmstadt, Germany). The matrix for the phylogenetic analysis comprises the cytochrome b sequences of N. pygmaeus and those of 44 other bovid species downloaded from GenBank (see Supplementary material 1). All these sequences were previously used in published phylogenetic analyses, and were carefully checked against other available GenBank sequences to avoid using sequences from misidentified individuals. The phylogenetic was conducted with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck, 2003) with the three-codon positions set as separate partitions. We used a GTR + GAMMA model for codon positions 1 and 3, a HKY + GAMMA model for codon position 2 (selected using hierarchical likelihood ratio tests with MrModeltest 2.3 (Nylander, 2004)), and default priors. We conducted two independent runs with 5 million generations each, sampling every 1000th generation. The standard deviation of split frequencies <0.01 was used as an indication for convergence; the first 1 million generations were discarded as burn-in. Parameter convergence and mixing were monitored with Tracer v. 1.5 (Rambaut and Drummond, 2007). An additional Maximum Parsimony analysis was conducted with PAUP* (Swafford, 2003) using a heuristic search with 5000 random-addition replicates. The Bootstrap analysis consisted of 1000 pseudoreplicates, each with 100 random-addition replicates. All characters were equally weighted. Alignments, MrBayes and PAUP* commands, the consensus tree of the Bayesian tree samples and the single most-parsimonious tree are available in Supplementary material 1. For the morphometric analyses, 95 skulls including N. pygmaeus (11 specimens), N. moschatus (15), N. batesi (10), Oreotragus oreotragus (15), Philantomba monticola (11), Sylvicapra grimmia (16), Cephalophus rufilatus (8), and Cephalophus silvicultor (9) (for museum catalogue numbers see Supplementary material 2) were measured in the Museum für Naturkunde in Berlin, the Natural History Museum in London, the Zoologische Staatssammlung München, and the University Museum of Zoology Cambridge. For each skull, 23 measurements were included (this excludes horn measurements, as the females of Neotragus and of several other species are hornless). Missing values were substituted by the average value of all specimens belonging to the same species (and sex if known). The original measurement data (including horn measurements) are available in Supplementary material 2; measurements were taken as depicted in Fig. 2 in Bärmann et al. (2013b). Measurements were log 10-transformed prior to analysis to account for size differences between the included species. Principal component analysis and discriminant analysis were carried out with IBM SPSS statistics 19. The phylogenetic analyses clearly support the polyphyly of the old tribe “Neotragini”. Moreover, polyphyly was found for the genus Neotragus itself (Fig. 1C and Supplementary material 1). In both analyses, Bayesian Inference (BI) and Maximum Parsimony (MP), N. batesi and N. moschatus were sister species (Posterior Probability (PP) = 1, Bootstrap support = 80). The BI analysis placed both together as the sister taxon of Aepyceros (PP = 0.66), while N. pygmaeus was resolved as closely related to Oreotragus + Cephalophini (PP = 1). The MP analysis placed N. batesi + N. moschatus as the sister
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taxon to all remaining Antilopinae (no Bootstrap support), whereas N. pygmaeus was the sister species of the dwarf antelope Dorcatragus megalotis (no Bootstrap support). The principal component analysis including N. pygmaeus, the two Nesotragus species, Oreotragus, and four species of Cephalophini shows almost complete species separation by principal components 1 and 2 (together accounting for 94% of the data variance). Component 1 is mainly a size component, whereas component 2 primarily accounts for differences in the size of the auditory bullae, the width of the skull, and the distance of the supraorbital foramina. The analysis shows an intermediate position of N. pygmaeus between the two Nesotragus species on the one hand and Cephalophini on the other (Fig. 1D). A discriminant analysis with three pre-defined groups (Cephalophini, Oreotragus, and Nesotragus) shows no overlap of N. pygmaeus with any of these groups (Fig. 1E). When forced to assign the N. pygmaeus specimens to one of the pre-defined groups, five specimens are assigned to Cephalophini and six specimens to Nesotragus. Neither the molecular analyses of cytochrome b sequences nor the morphometric analyses using skull measurements show a close relationship of N. pygmaeus with the other two “Neotragus” species, N. moschatus and N. batesi. The phylogenetic analyses place the latter two as the sister group of the remaining Antilopinae (with or without Aepyceros resolved as close relative) while N. pygmaeus is resolved as nested deeply within the Antilopinae, possibly as close relative of Oreotragus and the Cephalophini (result from BI). Of course, these results from the analyses of only one single mitochondrial gene should be re-evaluated using nuclear gene sequences. The close relationship of Oreotragus and Cephalophini that is commonly found in analyses of mitochondrial genes (e.g., Hassanin et al., 2012) does not always show up in nuclear gene analyses, where Oreotragus is sometimes placed as sister taxon to Reduncini, (Bärmann et al., 2013a). The position of N. pygmaeus might therefore change when more data are included. New sequences from N. pygmaeus might even help further resolving the phylogeny of Bovidae by breaking up long branches. In any case, the genus Neotragus, as it was commonly defined, is clearly polyphyletic; N. moschatus and N. batesi are not congeneric with Neotragus pygmaeus. We therefore suggest resurrecting the genus Nesotragus von Dueben, 1846 for N. moschatus and N. batesi. It has priority over the name Hylarnus that was erected by Thomas (1906) for his new species H. harrisoni, a synonym of N. batesi. These new results provide yet another example for polyphyly in dwarf antelopes. Previous authors have excluded Oreotragus, Ourebia, Dorcatragus, Madoqua and Raphicerus from Neotragini (e.g., Gentry, 1992; Hassanin and Douzery, 1999; Matthee and Davis, 2001; Ropiquet et al., 2009; Hassanin et al., 2012; Schikora, 2012; Bärmann et al., 2013a), and now only a single species is left in this group: the type species Neotragus pygmaeus itself. It is still unknown whether the similarity of the dwarf species, that for such a long time precluded their true phylogenetic relationships, is due to shared plesiomorphic characters that stem from a smallbodied common ancestor of all living Antilopinae, or whether it derives from convergence due to repeated miniaturisation in different clades of Bovidae. This is something worth investigating using ancestral-states-reconstruction analyses in the future.
Acknowledgements We thank the following museums for giving us access to specimens in their collections: Museum für Naturkunde, Berlin (F. Mayer and N. Lange), Natural History Museum, London (R. Portela Miguez), University Museum of Zoology, Cambridge (R. Asher and M. Lowe) and Zoologische Staatssammlung, München (R. Kraft and M. Hiermeier). We also thank Colin Groves and two anonymous reviewers
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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for their comments that helped improving the manuscript. The study is based on data collected during the doctoral studies of both authors. E.V.B. was funded by the Natural Environment Research Council (grant number PFAG/027 task 2), the Cambridge European Trust and the Balfour Fund of the Department of Zoology in Cambridge; T.S. was funded by Hesse’s Ministry of Higher Education, Research, and the Arts (funding programme “LOEWE–Landes Offensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz”).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mambio. 2014.01.001. References Ansell, W.F.H., 1972. Part 2: 15 Family Artiodactyla. In: Meester, J., Setzer, H.W. (Eds.), The Mammals of Africa: An Identification Manual. Smithsonian Institution Press, Washington, DC, pp. 1–84. Arnason, U., Gullberg, A., 1996. Cytochrome b nucleotide sequences and the identification of five primary lineages of extant cetaceans. Mol. Biol. Evol. 13, 407–417. Bärmann, E.V., Rössner, G.E., Wörheide, G., 2013a. A revised phylogeny of Antilopini (Bovidae, Artiodactyla) using combined mitochondrial and nuclear genes. Mol. Phylogenet. Evol. 67, 484–493. Bärmann, E.V., Wronski, T., Lerp, H., Azanza, B., Erpenbeck, D., Rössner, G.E., Wörheide, G., 2013b. A morphometric and genetic framework for the genus Gazella de Blainville 1816 with special focus on Arabian and Levantine Mountain gazelles. Zool. J. Linn. Soc., http://dx.doi.org/10.1111/zoj.12066. Birungi, J., Roy, M.S., Arctander, P., 1998. DMSO-preserved samples as a source of mRNA for RT-PCR. Mol. Ecol. 7, 1429–1430. Gentry, A.W., 1992. The subfamilies and tribes of the family Bovidae. Mamm. Rev. 22 (1), 1–32. Groves, C.P., Grubb, P., 2011. Ungulate Taxonomy. Johns Hopkins University Press, Baltimore.
Haltenorth, T., 1963. Klassifikation der Säugetiere, 18. Ordnung Paarhufer Artiodactyla. Handbuch der Zoologie Band VIII: Mammalia. de Gruyter, München, pp. 1–167. Hassanin, A., Douzery, E.J.P., 1999. The tribal radiation of the family Bovidae (Artiodactyla) and the evolution of the mitochondrial cytochrome b gene. Mol. Phylogenet. Evol. 13 (2), 227–243. Hassanin, A., Ropiquet, A., Couloux, A., Cruaud, C., 2009. Evolution of the mitochondrial genome in mammals living at high altitude: new insights from a study of the tribe Caprini (Bovidae, Antilopinae). J. Mol. Evol. 68 (4), 293–310. Hassanin, A., Delsuc, F., Ropiquet, A., Hammer, C., Jansen van Vuuren, B., Matthee, C., Ruiz-Garcia, M., Catzeflis, F., Areskoug, V., Nguyen, T.T., Couloux, A., 2012. Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes. C. R. Biol. 335, 32–50. IUCN Red List of Threatened Species, Version 2013.1, www.iucnredlist.org (downloaded 12.09.13). Matthee, C.A., Davis, S.K., 2001. Molecular insights into the evolution of the family Bovidae: a nuclear DNA perspective. Mol. Biol. Evol. 18 (7), 1220–1230. Nowak, R.M., 1999. Walker’s Mammals of the World. The Johns Hopkins University Press, Baltimore. Nylander, J.A.A., 2004. MrModeltest v2. Program Distributed by the Author. Evolutionary Biology Centre, Uppsala University. Pitra, C., Kock, R.A., Hofmann, R., Lieckfeldt, D., 1998. Molecular phylogeny of the critically endangered Hunter’s antelope (Beatragus hunteri Sclater 1889). J. Zool. Syst. Evol. Res. 36, 179–184. Rambaut, A., Drummond, A.J., 2007. Tracer v1.4, Available from: http:// beast.bio.ed.ac.uk/Tracer Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Ropiquet, A., Li, B., Hassanin, A., 2009. SuperTRI: a new approach based on branch support analyses of multiple independent data sets for assessing reliability of phylogenetic inferences. C. R. Biol. 332 (9), 832–847. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York. Schikora, T., (Dissertation) 2012. Climate-linked temporal and spatial patterns in the evolution of African bovids. Goethe-Universität Frankfurt am Main. Swafford, D.L., 2003. PAUP*: Phylogenetic Analysis Using Parsimony *and Other Methods. Version 4. Sinauer Associates, Sunderland, MA. Thomas, H., 1906. On a new pygmy antelope obtained by Col. J. J. Harrison in the Semliki forest. Ann. Mag. Nat. Hist. 18 (7), 149. Xu, S.Q., Yang, Y.Z., Zhou, J., Jing, G.E., Chen, Y.T., Wang, J., Yang, H.M., Wang, J., Yu, J., Zheng, X.G., Ge, R.L., 2005. A mitochondrial genome sequence of the Tibetan antelope (Pantholops hodgsonii). Genomics Proteomics Bioinform. 3 (1), 5–17.
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
ARTICLE IN PRESS Mammalian Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Mammalian Biology journal homepage: www.elsevier.com/locate/mambio
Short Communication
The polyphyly of Neotragus – Results from genetic and morphometric analyses Eva V. Bärmann a,b,∗ , Tim Schikora c,d a
Department of Zoology, University of Cambridge, Downing Street, CB2 3EJ, UK Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Invalidenstrasse 43, 10115 Berlin, Germany Biodiversität und Klima Forschungszentrum, 60325 Frankfurt am Main, Germany d Zoo Dortmund, Mergelteichstr. 80, 44225 Dortmund, Germany b c
a r t i c l e
i n f o
Article history: Received 24 September 2013 Accepted 6 January 2014 Handled by Paul Grobler Available online xxx Keywords: Neotragus Cytochrome b Principal component analysis Discriminant analysis Skull morphology
a b s t r a c t Dwarf antelope species were commonly united in the tribe “Neotragini” (Bovidae, Mammalia) due to their general morphological appearance. However, phylogenetic analyses have shown that not all dwarf antelopes are closely related, so it was suggested to restrict the name Neotragini to the type genus Neotragus. In our study we use mitochondrial cytochrome b sequences and linear skull measurements to further investigate the similarity of all three Neotragus species. Our analyses support the close relationship of N. moschatus and N. batesi. However, N. pygmaeus – the type species, which was never before included in phylogenetic analyses – is not closely related. It might share a most recent common ancestor with another “dwarf antelope”, the Klipspringer Oreotragus oreotragus, and the duikers in the taxon Cephalophini. Hence, we suggest resurrecting the genus Nesotragus von Dueben, 1846 for Nesotragus moschatus and N. batesi. © 2014 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.
The genus Neotragus C. H. Smith, 1827 comprises the smallest antelopes alive today, with body mass as little as 2 kg in the Royal antelope, Neotragus pygmaeus (Linnaeus, 1758) (Groves and Grubb, 2011). Along with this type species usually two more species are included in Neotragus: the Suni, N. moschatus (von Dueben, 1846), and Bates’ pygmy antelope, N. batesi De Winton, 1903. All three species have an allopatric distribution and inhabit tropical forests of West, Central and East Africa (see Fig. 1A). The Suni was originally placed in its own genus Nesotragus von Dueben, 1846, and later synonymised with Neotragus (Ansell, 1972). Bates’ pygmy antelope on the other hand was usually regarded as congeneric with N. pygmaeus, although Haltenorth (1963) in his “Classification of Mammals” referred it to Nesotragus. Historically, Neotragus was united with other small antelopes like the Klipspringer Oreotragus oreotragus (Zimmermann, 1783), Oribi Ourebia ourebi (Zimmermann, 1783), Beira Dorcatragus megalotis Menges, 1894, the Dikdiks Madoqua spp. Ogilby, 1836 and the Steen- and Grysboks Raphicerus spp. C. H. Smith, 1827 in a group called “Neotragini” (see Nowak 1999). They all have some morphological features in common, like short and straight horns, and show
∗ Corresponding author at: Museum für Naturkunde, Leibniz-Institut für Evolutions- und Biodiversitätsforschung, Invalidenstrasse 43, 10115 Berlin, Germany. Tel.: +49 0179 2416608. E-mail address: [email protected] (E.V. Bärmann).
a similar social organisation where one male and usually one female share a permanent territory. However, morphological and molecular analyses of the past 20 years have shown that “Neotragini” in the traditional configuration is a polyphyletic assemblage (e.g., Gentry, 1992; Hassanin and Douzery, 1999; Matthee and Davis, 2001; Ropiquet et al., 2009; Hassanin et al., 2012; Schikora, 2012; Bärmann et al., 2013a): Ourebia, Raphicerus, Madoqua, and Dorcatragus are now included in Antilopini, whereas Oreotragus is often placed in its own tribe Oreotragini with yet unresolved phylogenetic relationships. So at present only the genus Neotragus remains in Neotragini. Both N. moschatus and N. batesi are characterised by mitochondrial DNA sequences. In their recent molecular analysis Hassanin et al. (2012) confirmed that they are sister-species, and are presumably closely related to the Impala, Aepyceros melampus (Lichtenstein, 1812). Both these Neotragus species and Aepyceros also share a unique morphological character: a fenestra between premaxillary and maxillary (see Fig. 1B). However, this fenestra is absent in N. pygmaeus. We sequenced the mitochondrial cytochrome b gene of N. pygmaeus in order to investigate its phylogenetic relationship with the other two Neotragus species (in the following called Nesotragus). In addition, we used linear skull measurements in multivariate morphometric analyses to further study the similarity of N. pygmaeus, N. moschatus, N. batesi, O. oreotragus, and four species of Cephalophini.
1616-5047/$ – see front matter © 2014 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.mambio.2014.01.001
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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Fig. 1. (A) Geographic ranges of Neotragus pygmaeus, N. batesi, and N. moschatus in Africa (map re-drawn from IUCN Red List of threatened species www.iucnredlist.org, September 2013); (B) skulls of N. pygmaeus (male, ZSM 1963-143), N. batesi (female, ZSM 1975-28), and N. moschatus (male, ZMB MAM 57144), the fenestra between premaxillary and maxillary in N. batesi and N. moschatus (marked with arrows) is absent in N. pygmaeus; (C) consensus tree from 8000 post burn-in trees of Bayesian analysis of cytochrome b sequences, branch labels = posterior probabilities, Neotragus and Nesotragus are highlighted with bold letters and thick branches, lower case letters correspond to publication references of the sequences: (a) Hassanin et al. (2012), (b) Hassanin et al. (2009), (c) Pitra et al. (1998), (d) Xu et al. (2005), (e) Birungi et al. (1998), and (f) Tungsudjai et al. (no journal reference); (D) bi-variate plot of specimen scores for the two main components in a principal component analysis (PCA) of 23 linear skull measurements; (E) bi-variate plot of specimen scores for two discriminant functions in a discriminant analysis (DA) of 23 linear skull measurements, the discriminant functions derived from the three pre-defined groups Nesotragus, Cephalophini, and Oreotragus are used for classification of N. pygmaeus specimens.
A full blood sample of N. pygmaeus was collected by T.S. from a specimen caught by a local hunter in Ghana (West of Accra, South of Dunkwa Village, along the Kumasi – Yamoransa Rd, GPS coordinates: 5.28838904◦ N, −1.21371042◦ E). It was conserved in 2 vol. ethanol (90%) and stored as cool as possible. DNA was extracted after washing with 500 l CTAB-buffer and overnight incubation
with 20 l proteinase K (10 mg/ml) and 0.65 l -mercaptoethanol at 56 ◦ C, using standard phenol–chloroform extraction (Sambrook and Russell, 2001). The complete cytochrome b sequence (1140 bp) was amplified successfully with the primer pair CytbH and CytbL (Arnason and Gullberg, 1996). The PCR reactions were performed using 0.625 U peqGOLD Taq-DNA-Polymerase (PEQLAB
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s recommendations and performed according to the following protocol: initial denaturation at 94 ◦ C for 3 min, 30 cycles denaturation at 94 ◦ C, annealing at 45 ◦ C, and extension at 72 ◦ C (each step 1 min), and final extension at 72 ◦ C for 10 min. Following the manufacturer’s instructions the PCR products were purified using Sephadex G50 (Sigma–Aldrich, Munich, Germany). The fragments were amplified in forward and in reverse direction in two separate sequencing-reactions using the primers CytbH and CytbL. After ethanol precipitation, sequencing was performed using BigDyeTerminator v.3 on an ABI 3730 DNA Analyzer (Applied Biosystems, Darmstadt, Germany). The matrix for the phylogenetic analysis comprises the cytochrome b sequences of N. pygmaeus and those of 44 other bovid species downloaded from GenBank (see Supplementary material 1). All these sequences were previously used in published phylogenetic analyses, and were carefully checked against other available GenBank sequences to avoid using sequences from misidentified individuals. The phylogenetic was conducted with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck, 2003) with the three-codon positions set as separate partitions. We used a GTR + GAMMA model for codon positions 1 and 3, a HKY + GAMMA model for codon position 2 (selected using hierarchical likelihood ratio tests with MrModeltest 2.3 (Nylander, 2004)), and default priors. We conducted two independent runs with 5 million generations each, sampling every 1000th generation. The standard deviation of split frequencies <0.01 was used as an indication for convergence; the first 1 million generations were discarded as burn-in. Parameter convergence and mixing were monitored with Tracer v. 1.5 (Rambaut and Drummond, 2007). An additional Maximum Parsimony analysis was conducted with PAUP* (Swafford, 2003) using a heuristic search with 5000 random-addition replicates. The Bootstrap analysis consisted of 1000 pseudoreplicates, each with 100 random-addition replicates. All characters were equally weighted. Alignments, MrBayes and PAUP* commands, the consensus tree of the Bayesian tree samples and the single most-parsimonious tree are available in Supplementary material 1. For the morphometric analyses, 95 skulls including N. pygmaeus (11 specimens), N. moschatus (15), N. batesi (10), Oreotragus oreotragus (15), Philantomba monticola (11), Sylvicapra grimmia (16), Cephalophus rufilatus (8), and Cephalophus silvicultor (9) (for museum catalogue numbers see Supplementary material 2) were measured in the Museum für Naturkunde in Berlin, the Natural History Museum in London, the Zoologische Staatssammlung München, and the University Museum of Zoology Cambridge. For each skull, 23 measurements were included (this excludes horn measurements, as the females of Neotragus and of several other species are hornless). Missing values were substituted by the average value of all specimens belonging to the same species (and sex if known). The original measurement data (including horn measurements) are available in Supplementary material 2; measurements were taken as depicted in Fig. 2 in Bärmann et al. (2013b). Measurements were log 10-transformed prior to analysis to account for size differences between the included species. Principal component analysis and discriminant analysis were carried out with IBM SPSS statistics 19. The phylogenetic analyses clearly support the polyphyly of the old tribe “Neotragini”. Moreover, polyphyly was found for the genus Neotragus itself (Fig. 1C and Supplementary material 1). In both analyses, Bayesian Inference (BI) and Maximum Parsimony (MP), N. batesi and N. moschatus were sister species (Posterior Probability (PP) = 1, Bootstrap support = 80). The BI analysis placed both together as the sister taxon of Aepyceros (PP = 0.66), while N. pygmaeus was resolved as closely related to Oreotragus + Cephalophini (PP = 1). The MP analysis placed N. batesi + N. moschatus as the sister
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taxon to all remaining Antilopinae (no Bootstrap support), whereas N. pygmaeus was the sister species of the dwarf antelope Dorcatragus megalotis (no Bootstrap support). The principal component analysis including N. pygmaeus, the two Nesotragus species, Oreotragus, and four species of Cephalophini shows almost complete species separation by principal components 1 and 2 (together accounting for 94% of the data variance). Component 1 is mainly a size component, whereas component 2 primarily accounts for differences in the size of the auditory bullae, the width of the skull, and the distance of the supraorbital foramina. The analysis shows an intermediate position of N. pygmaeus between the two Nesotragus species on the one hand and Cephalophini on the other (Fig. 1D). A discriminant analysis with three pre-defined groups (Cephalophini, Oreotragus, and Nesotragus) shows no overlap of N. pygmaeus with any of these groups (Fig. 1E). When forced to assign the N. pygmaeus specimens to one of the pre-defined groups, five specimens are assigned to Cephalophini and six specimens to Nesotragus. Neither the molecular analyses of cytochrome b sequences nor the morphometric analyses using skull measurements show a close relationship of N. pygmaeus with the other two “Neotragus” species, N. moschatus and N. batesi. The phylogenetic analyses place the latter two as the sister group of the remaining Antilopinae (with or without Aepyceros resolved as close relative) while N. pygmaeus is resolved as nested deeply within the Antilopinae, possibly as close relative of Oreotragus and the Cephalophini (result from BI). Of course, these results from the analyses of only one single mitochondrial gene should be re-evaluated using nuclear gene sequences. The close relationship of Oreotragus and Cephalophini that is commonly found in analyses of mitochondrial genes (e.g., Hassanin et al., 2012) does not always show up in nuclear gene analyses, where Oreotragus is sometimes placed as sister taxon to Reduncini, (Bärmann et al., 2013a). The position of N. pygmaeus might therefore change when more data are included. New sequences from N. pygmaeus might even help further resolving the phylogeny of Bovidae by breaking up long branches. In any case, the genus Neotragus, as it was commonly defined, is clearly polyphyletic; N. moschatus and N. batesi are not congeneric with Neotragus pygmaeus. We therefore suggest resurrecting the genus Nesotragus von Dueben, 1846 for N. moschatus and N. batesi. It has priority over the name Hylarnus that was erected by Thomas (1906) for his new species H. harrisoni, a synonym of N. batesi. These new results provide yet another example for polyphyly in dwarf antelopes. Previous authors have excluded Oreotragus, Ourebia, Dorcatragus, Madoqua and Raphicerus from Neotragini (e.g., Gentry, 1992; Hassanin and Douzery, 1999; Matthee and Davis, 2001; Ropiquet et al., 2009; Hassanin et al., 2012; Schikora, 2012; Bärmann et al., 2013a), and now only a single species is left in this group: the type species Neotragus pygmaeus itself. It is still unknown whether the similarity of the dwarf species, that for such a long time precluded their true phylogenetic relationships, is due to shared plesiomorphic characters that stem from a smallbodied common ancestor of all living Antilopinae, or whether it derives from convergence due to repeated miniaturisation in different clades of Bovidae. This is something worth investigating using ancestral-states-reconstruction analyses in the future.
Acknowledgements We thank the following museums for giving us access to specimens in their collections: Museum für Naturkunde, Berlin (F. Mayer and N. Lange), Natural History Museum, London (R. Portela Miguez), University Museum of Zoology, Cambridge (R. Asher and M. Lowe) and Zoologische Staatssammlung, München (R. Kraft and M. Hiermeier). We also thank Colin Groves and two anonymous reviewers
Please cite this article in press as: Bärmann, E.V., Schikora, T., The polyphyly of Neotragus – Results from genetic and morphometric analyses. Mammal. Biol. (2014), http://dx.doi.org/10.1016/j.mambio.2014.01.001
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for their comments that helped improving the manuscript. The study is based on data collected during the doctoral studies of both authors. E.V.B. was funded by the Natural Environment Research Council (grant number PFAG/027 task 2), the Cambridge European Trust and the Balfour Fund of the Department of Zoology in Cambridge; T.S. was funded by Hesse’s Ministry of Higher Education, Research, and the Arts (funding programme “LOEWE–Landes Offensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz”).
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