Research Article Cytochrome c Oxidase Sequences of Zambian ...downloads.hindawi.com/journals/ijz/2016/1808912.pdf · License, which permits unrestricted use, distribution, and reproduction

Research ArticleCytochrome c Oxidase Sequences of Zambian Wildlife Helps toIdentify Species of Origin of Meat

Michelo Syakalima1 Musso Munyeme2 and Jun Yasuda3

1Faculty of Agriculture Science and Technology Centre for Animal Health North West University Mafikeng CampusPrivate Bag Box X2046 Mmabatho 2735 South Africa2School of Veterinary Medicine University of Zambia PO Box 32379 Lusaka Zambia3School of Agricultural Sciences Iwate University Morioka Japan

Correspondence should be addressed to Michelo Syakalima michelosyakalimanwuacza

Received 15 July 2016 Revised 22 October 2016 Accepted 27 October 2016

Academic Editor Marco Cucco

Copyright copy 2016 Michelo Syakalima et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Accurate species identification is a crucial tool in wildlife conservation Enforcement of antipoaching law is more achievable withrobust molecular identification of poached meat Determining the region where the animal may have been taken from would alsobe a useful tool in suppression of cross-border trade of poached meat We present data from a cytochrome c oxidase ldquobarcodingrdquostudy of Zambian ruminants that adequately identifies the species of origin ofmeat samples Furthermore themethod demonstratespossible improvement and application in regional variation in sequence identity that has a potential for discriminatingmeat samplesfrom different subpopulations

1 Introduction

Marginal wildlife utilization has historically been an integralpart of most African cultures Unfortunately recent trendshave shown that this has now been transformed from sub-sistence into an illegal multimillion dollar industry [1 2]with often negative consequences on target animal speciesrsquopopulations and the environment

In Zambia poaching for game meat or trade in wildlifeproducts is one of the most damaging human activities towild animal populations Poachers evade law enforcement byremoving easily recognisable body parts when transportinggame meat or by processing or semiprocessing meat to makeit difficult to identify the species from which it was derivedAfter such treatment commonly used identificationmethodssuch as smell or meat texture appearance lack scientificcredibility and cannot be used during prosecution

Wildlife forensics often provides scientific credibility inprosecuting wildlife crime This study is based upon the useof DNA sequence analysis of meat or other animal tissuesto ensure accurate species identification The most commonmethods of species identification use mitochondrial DNA

particularly the cytochrome b (Cytb) and cytochrome oxi-dase I (COI) [2ndash5] genesTheCOI gene has been identified bythe Barcode of Life Project as the best gene for differentiatinganimal species A broad range of primers are available forthis gene which are applicable to a wide range of taxa [6 7]The sequence of the gene is relatively short and changes tothe sequence occur more slowly than in the Cytb gene [8]The COI gene can be used to characterize individuals notonly at higher taxonomic levels (phyla and order) but alsoat a species level [9 10] Through the Barcode of Life Project(httpwwwbarcodinglifeorg) the COI gene is the focus ofaworldwidemolecular effort to characterize all living animalsusing a single genetic entity [11] The COI gene thereforepotentially allows forensic scientists to identify the species oforigin of biological samples through DNA sequence analysisand comparison with the Barcode of Life Database (BOLD)This database is still being built but it will be possibleto identify the species from which a sample is originatedif the species has already been deposited in the databaseThe presence of many sequences from different regions alsohas potential to help in analysing geographical clustering ofbarcoding sequences which can be very useful for bushmeat

Hindawi Publishing CorporationInternational Journal of ZoologyVolume 2016 Article ID 1808912 6 pageshttpdxdoiorg10115520161808912

2 International Journal of Zoology

Table 1 The species number of samples and the method of sample preservation used in this study

Common name Scientific name Animals sampled Sample preservationPuku Kobus vardoni 7 70 ETOHEland Taurotragus oryx 3 70 ETOHImpala Aepyceros melampus 3 70 ETOHKafue Lechwe Kobus leche kafuensis 3 70 ETOH amp PBSBushbuck Tragelaphus sylvaticus 2 PBS and frozenWaterbuck Kobus ellipsiprymnus 2 PBS and frozenBuffalo Syncerus caffer 2 PBS and frozenWildebeest Connochaetes taurinus 2 PBS and frozenSable Hippotragus niger 2 PBS and frozenReedbuck Redunca arundinum 2 FormalinHartebeest Alcelaphus buselaphus 1 FormalinETOH ethanol PBS phosphate buffered saline

identification and tracing A preliminary validation study hasalready been conducted against forensic standards [12] butthe technique still needs to be tested in different parts of theworld for both identification and geographical clustering ofbarcoding sequences [13]

This study focused on the application of this techniqueto some Zambian wildlife and comparison of the sequencesobtained by COI barcoding to previously characterizedspecies from elsewhere in Africa Several common gamespecies for which there was no previous sequence identifica-tion available in the publicly accessible databases were alsocharacterized and deposited

2 Materials and Methods

21 Study Samples Tissue samples were collected from 29known individual animals representing 11 species of Bovidaein game ranches and national parks around Lusaka and southLuangwa Zambia The samples were collected with the helpof wildlife police officers and game ranch staff to guaranteespecies identification Table 1 shows the species sampled thenumber of animals sampled for each species and themethodsused to preserve samples before DNA extraction

22 DNA Extraction DNA was extracted using the QiagenBlood and Tissue Kit according to standard protocols for allsamples except those preserved in formalin where the QiagenFFPE kit was used

23 Polymerase Chain Reaction and Sequencing The COIgene fragments were amplified using the polymerase chainreactions (PCR) in 25 120583L reaction mixture The primers usedwere COIbF (51015840-TTTCAACCAACCACAAAGACATCGG-31015840) and COIbR (51015840-TATACTTCAGGGTGTCCAAAGAAT-CA-31015840) [13] Amplification was carried out in 10x reactionbuffer containing 25mM of MgCl

2 02mM each dNTP and

075 units of Taq DNA polymerase (All New England Bio-labs httpswwwnebcom) 10mMeachprimer (Invitrogenwwwinvitrogencom) and 1 uL template DNA PCR ampli-fication was performed on Techne TC-512 Thermal Cycler(Barloworld Scientific Ltd) with 15min at 95∘C for initial

polymerase activation followed by 35 cycles of amplification(30 s at 94∘C 30 s at 55∘C and 1min at 72∘C) and finalextension for 10min at 72∘C Products were viewed via gelelectrophoresis with ethidium bromide and amplified PCRproducts were purified using a kit (Nucleospin Extract IIMacherey-Nagel) Sequencing was performed using BigDyeterminator v31 as per protocol on an ABI 3730 DNAsequencer (Applied Biosystems)

24 Data Analysis Bidirectional contig assembly and editingwere carried out using the CAP3 contig assembly pro-gram [14] through BIOEDIT [15] Species identificationwas performed via BLASTn [16] searches against the NCBInucleotide collection and BOLD databases

Species identifications were verified using a phylogeneticdataset consisting of 27 sequences representingmost subfam-ilies of Bovidae present in Africa Where available sequencesfrom southern or central African isolates were used Acces-sion numbers and geographic origins of these sequences areshown in Supplementary Table 1 in Supplementary Mate-rial available online at httpdxdoiorg10115520161808912Sequences were aligned using MUSCLE [17 18] under thedefault settings Phylogenetic trees were generated usingPhyML [18 19] under the general time reversible nucleotidesubstitutionmodelwith optimised tree topology and betweensite variations Branch support was calculated throughPhyML using the approximate likelihood ratio test [19]A sequence from the deer (Dama dama) was used as anoutgroup

After species identification phylogenetic trees were gen-erated to establish whether barcoding information is suffi-cient to establish the geographic origin of samples A datasetwas created with all available COI sequences of the testspecies set A phylogenetic tree for this dataset was generatedas above The sequences included in this analysis are listed inSupplementary Table 2

3 Results

Of the 11 species examined the COI genes of 9 species wereamplified successfully The most representative sequence of

International Journal of Zoology 3

Aepycerotinae 1

1

085

099

099

1083

076 098

08

09

091

077078

1

1093

094096

092083

092086

079

Bovinae

Antilopinae

Alcelaphinae

Hartlebeest Cephalophinae

Hippotraginae

Caprinae

Reduncinae

Reedbuck Peleinae

minus

DeerIMPALA

ImpalaCowZebu

BongoSitatunga

BUSHBUCKBushbuck

Greater Kudu

Lesser Kudu

NyalaELAND

Common ElandGiant Eland

BUFFALOBuffalo

Black WildebeestWildebeest

SassabieWILDEBEEST

OryxAddax

SABLESable

GoatSheep

Roan Antelope

WATERBUCKWaterbuck

KAFUE LECHWEPUKU

Kafue Lechwe

Figure 1 PhyML maximum likelihood phylogenetic tree of the cytochrome c oxidase subunit 1 gene for waterbuck impala reedbuck oryxaddax duiker sheep goat nyala sitatunga bongo hartebeest sassabies cattle deer kudu wildebeest eland bushbuck and buffalo sequencesZambian test sequences are shown in capital letters (SupplementaryTable 1) Bootstrap values exceeding 75are shownon the nodesThedeersequence was used as an outgroup to root the tree GenBank accession numbers and geographical information are provided in SupplementaryTable 1

each of these species was deposited in the GenBank databasewith the following reference numbers puku (Kobus var-doni) JQ690383 eland (Taurotragus oryx) JQ690384 impala(Aepyceros melampus) JQ690386 Kafue Lechwe (Kobusleche kafuensis) JQ690387 waterbuck (Kobus ellipsiprymnus)JQ690391 buffalo (Syncerus caffer) JQ690392 wildebeest(Connochaetes taurinus) JQ690393 sable (Hippotragus niger)JQ690394 and bushbuck (Tragelaphus scriptus) JQ690389No PCR products were recovered from reedbuck (Reduncaarundinum) or Lichtensteinrsquos hartebeest (Alcelaphus busela-phus lichtensteini)

All nine of these species could be robustly identifiedby similarity searching or phylogenetic analysis The BOLDdatabase has sequences for all the test species except puku Allother samples could be identified robustly at a species levelwith the exception of the wildebeest which was only resolved

to genus level and bushbuck which was not recognisedUsing BLASTn against the nr database which is slightlyless stringent bushbuck was identified at species level andwildebeest and puku at genus level In phylogenetic analysisall samples with an available African barcode sequenceclustered closely and robustly with their species of origin asshown by the values in Figure 1

In the geographic analysis (Figure 2) there was noconsistent relationship between country of origin and phy-logeny For impala and wildebeest sequences tended tocluster by country For buffalo bushbuck waterbuck andeland however several clades contained sequences of mixedorigin including some geographically distant sequencessuch as waterbuck sequences from Tanzania and NigerZambian sequences were generally fairly distinct fromsequences from elsewhere in Africa Insufficient sequences


N

E

K

T

ZAM

Z B

SA

T1

T10

T12T4

T9

T15

T5

T7T3

N1

T2

T2

T7

T2

T1

T1

T5T8

T6

T4T3

T9

T6

T5 T1

T4

T3

T2

T3

K14T4

T1

T2 T11E2

E4

E3

B9Z5B5Z6

E5E6

T3K12

K13

K15K16

K17

T10

T10

T1

T6 T2

T3

T4T11

T13

T14ZAM

ZAM

ZAM

Impala

Eland

Bushbuck

SA B3

B2Buffalo

SA1

ZAM

Waterbuck

ZAM

ZAM

K18E1B1

E7Z3Z2Z1T5T8T9T7K19

Z4

B8B7

B6

Z7K20 B4

Wildebeest

Figure 2 PhyML maximum likelihood phylogenetic tree for available COI sequences from waterbuck impala wildebeest eland bushbuckand buffalo of known geographic origin Sequences are labelled as follows Zambia ZAM Ethiopia E Kenya K Tanzania T Zimbabwe ZBotswana B South Africa SA Niger N GenBank accession numbers are provided in Supplementary Table 2


were available for geographic analysis of the remainingsamples

4 Discussion

The basic technique of COI barcoding proved a robust meansof identification of meat samples from the species examinedOur findings here were in broad agreement with that ofBitanyi et al [13] in Tanzania that covered an overlappingrange of species confirming the applicability of this method-ology in forensic identification and conservation genetics ofgame species in general [20] The only species for whichsequence data could not be obtained were for those speci-mens preserved in formalin which has a known detrimentaleffect on DNA quality [21] We therefore concluded thatusing formalin to preserve samples may not be ideal for thismethodwhich is unfortunate becausemost samples collectedfrom the Zambian wild are preserved in formalin

Two species wildebeest and puku could only be iden-tified at a genus rather than species level For puku this issimply the result of a lack of data as no previous puku barcodesequences were available at the time of the studyThe barcod-ing technique was not suitable for very detailed identificationof samples if the species had not been sequenced previouslyHowever phylogenetic analysis showed that the sample wasof a Kobus sequence and not K ellipsiprymnus or K lechewhich only leaves four possible species and is likely to beadequate for most purposes This problem will become lesscommon as the barcoding database grows For wildebeestthe ambiguity was between two very closely related speciesthe blue wildebeest C taurinus and black wildebeest C gnouThe COI gene may be too conserved to distinguish betweenvery similar species although again the technique is likely tobe sufficient for most purposes when prosecuting poachingcases

The geographic analysis showed that the COI sequenceis generally not very reliable in determining the geographicorigin of a sample Some species wildebeest impala andeland did show distinct geographical clustering separatingTanzanian and Zambian sequences For buffalo bushbuckand waterbuck this was not the case Zambian sequencestended to fall outside of groups of sequences from otherAfrican countries and it would be worthwhile to sequencemore samples from these populations to see if they wouldform unique clusters All sequences analysed except thewaterbuck sequence from Niger were from adjacent coun-tries (Figure 2) so it is possible that the populations analysedfrom different countries are not isolated from each other It isalso likely that given the short length and high conservationof the COI gene there is too little genetic variation fordetailed within species analysis However it would generallybe possible to establish whether or not a sample is likely tohave come from a particular well characterized population

5 Conclusion

COI barcoding was consistently successful in easily identify-ing the species of origin of samples from Zambian wildlife

species providing that the species has been previously bar-coded When this was not the case samples could be identi-fied at genus level Therefore this technique would allowidentification of the origin of poached meat samples How-ever without significantly more data the technique cannotreliably establish which population of animals a sample isfrom

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The final part of this work was done with support fromthe Commonwealth Scholarship Commission Collection ofsamples and DNA extraction kits was supported by a Grant-in-Aid for Scientific Research (A) (21255010) from the JapanSociety for the Promotion of Science (JSPS) Special acknowl-edgements to Dr Rachael Tarlinton Dr Lisa Yon and DrRichard Emes for the molecular work and bioinformaticssupport Dr Rob Ogden and Lucy Webster for helping withthe PCR and general forensics requirements and Dr EmmaJelbert for advice on crime scene investigations

References

[1] P Ferrier ldquoThe economics of agricultural and wildlife smug-glingrdquo United States Development Agency Economic ResearchReport vol 8 pp 1ndash35 2009

[2] Interpol ldquoIllegal Ivory and Rhino horn trade targets of Interpolco-ordinated operation across Southern Africardquo httpwwwinterpolintNews-and-mediaNews-media-releases2010PR036

[3] C Bellis K J Ashton L Freney B Blair and L R GriffithsldquoA molecular genetic approach for forensic animal speciesidentificationrdquo Forensic Science International vol 134 no 2-3pp 99ndash108 2003

[4] H-M Hsieh L-H Huang L-C Tsai et al ldquoSpecies identifica-tion of rhinoceros horns using the cytochrome b generdquo ForensicScience International vol 136 no 1ndash3 pp 1ndash11 2003

[5] W Parson K Pegoraro H Niederstatter M Foger andM Steinlechner ldquoSpecies identification by means of thecytochrome b generdquo International Journal of LegalMedicine vol114 no 1-2 pp 23ndash28 2000

[6] O Folmer M Black W Hoeh R Lutz and R VrijenhoekldquoDNA primers for amplification of mitochondrial cytochromec oxidase subunit I from diverse metazoan invertebratesrdquoMolecular Marine Biology and Biotechnology vol 3 no 5 pp294ndash299 1994

[7] S K Verma and L Singh ldquoNovel universal primers establishidentity of an enormous number of animal species for forensicapplicationrdquo Molecular Ecology Notes vol 3 no 1 pp 28ndash312003

[8] M Lynch and P E Jarrell ldquoA method for calibrating molecularclocks and its application to animal mitochondrial DNArdquoGenetics vol 135 no 4 pp 1197ndash1208 1993

[9] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedings


of the Royal Society of London B Biological Sciences vol 270 no1512 pp 313ndash321 2003

[10] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Sciences vol 270 no 1 pp S96ndashS99 2003

[11] S Ratnasingham and P D N Hebert ldquobold the barcode of lifedata system (httpwwwbarcodinglifeorg)rdquoMolecular EcologyNotes vol 7 no 3 pp 355ndash364 2007

[12] N Dawnay R Ogden R McEwing G R Carvalho andR S Thorpe ldquoValidation of the barcoding gene COI foruse in forensic genetic species identificationrdquo Forensic ScienceInternational vol 173 no 1 pp 1ndash6 2007

[13] S Bitanyi G Bjoslashrnstad E M Ernest et al ldquoSpecies identifica-tion of Tanzanian antelopes using DNA barcodingrdquo MolecularEcology Resources vol 11 no 3 pp 442ndash449 2011

[14] X Huang and A Madan ldquoCAP3 a DNA sequence assemblyprogramrdquo Genome Research vol 9 no 9 pp 868ndash877 1999

[15] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[16] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[17] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[18] M Gouy S Guindon and O Gascuel ldquoSeaView version 4 amultiplatform graphical user interface for sequence alignmentand phylogenetic tree buildingrdquo Molecular Biology and Evolu-tion vol 27 no 2 pp 221ndash224 2010

[19] S Guindon and O Gascuel ldquoA simple fast and accurate algo-rithm to estimate large phylogenies by maximum likelihoodrdquoSystematic Biology vol 52 no 5 pp 696ndash704 2003

[20] R Ogden N Dawnay and R McEwing ldquoWildlife DNAforensicsmdashbridging the gap between conservation genetics andlaw enforcementrdquo Endangered Species Research vol 9 no 3 pp179ndash195 2009

[21] M Srinivasan D Sedmak and S Jewell ldquoEffect of fixatives andtissue processing on the content and integrity of nucleic acidsrdquoAmerican Journal of Pathology vol 161 no 6 pp 1961ndash19712002

Submit your manuscripts athttpwwwhindawicom

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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 The species number of samples and the method of sample preservation used in this study

Common name Scientific name Animals sampled Sample preservationPuku Kobus vardoni 7 70 ETOHEland Taurotragus oryx 3 70 ETOHImpala Aepyceros melampus 3 70 ETOHKafue Lechwe Kobus leche kafuensis 3 70 ETOH amp PBSBushbuck Tragelaphus sylvaticus 2 PBS and frozenWaterbuck Kobus ellipsiprymnus 2 PBS and frozenBuffalo Syncerus caffer 2 PBS and frozenWildebeest Connochaetes taurinus 2 PBS and frozenSable Hippotragus niger 2 PBS and frozenReedbuck Redunca arundinum 2 FormalinHartebeest Alcelaphus buselaphus 1 FormalinETOH ethanol PBS phosphate buffered saline

identification and tracing A preliminary validation study hasalready been conducted against forensic standards [12] butthe technique still needs to be tested in different parts of theworld for both identification and geographical clustering ofbarcoding sequences [13]

This study focused on the application of this techniqueto some Zambian wildlife and comparison of the sequencesobtained by COI barcoding to previously characterizedspecies from elsewhere in Africa Several common gamespecies for which there was no previous sequence identifica-tion available in the publicly accessible databases were alsocharacterized and deposited

2 Materials and Methods

21 Study Samples Tissue samples were collected from 29known individual animals representing 11 species of Bovidaein game ranches and national parks around Lusaka and southLuangwa Zambia The samples were collected with the helpof wildlife police officers and game ranch staff to guaranteespecies identification Table 1 shows the species sampled thenumber of animals sampled for each species and themethodsused to preserve samples before DNA extraction

22 DNA Extraction DNA was extracted using the QiagenBlood and Tissue Kit according to standard protocols for allsamples except those preserved in formalin where the QiagenFFPE kit was used

23 Polymerase Chain Reaction and Sequencing The COIgene fragments were amplified using the polymerase chainreactions (PCR) in 25 120583L reaction mixture The primers usedwere COIbF (51015840-TTTCAACCAACCACAAAGACATCGG-31015840) and COIbR (51015840-TATACTTCAGGGTGTCCAAAGAAT-CA-31015840) [13] Amplification was carried out in 10x reactionbuffer containing 25mM of MgCl

2 02mM each dNTP and

075 units of Taq DNA polymerase (All New England Bio-labs httpswwwnebcom) 10mMeachprimer (Invitrogenwwwinvitrogencom) and 1 uL template DNA PCR ampli-fication was performed on Techne TC-512 Thermal Cycler(Barloworld Scientific Ltd) with 15min at 95∘C for initial

polymerase activation followed by 35 cycles of amplification(30 s at 94∘C 30 s at 55∘C and 1min at 72∘C) and finalextension for 10min at 72∘C Products were viewed via gelelectrophoresis with ethidium bromide and amplified PCRproducts were purified using a kit (Nucleospin Extract IIMacherey-Nagel) Sequencing was performed using BigDyeterminator v31 as per protocol on an ABI 3730 DNAsequencer (Applied Biosystems)

24 Data Analysis Bidirectional contig assembly and editingwere carried out using the CAP3 contig assembly pro-gram [14] through BIOEDIT [15] Species identificationwas performed via BLASTn [16] searches against the NCBInucleotide collection and BOLD databases

Species identifications were verified using a phylogeneticdataset consisting of 27 sequences representingmost subfam-ilies of Bovidae present in Africa Where available sequencesfrom southern or central African isolates were used Acces-sion numbers and geographic origins of these sequences areshown in Supplementary Table 1 in Supplementary Mate-rial available online at httpdxdoiorg10115520161808912Sequences were aligned using MUSCLE [17 18] under thedefault settings Phylogenetic trees were generated usingPhyML [18 19] under the general time reversible nucleotidesubstitutionmodelwith optimised tree topology and betweensite variations Branch support was calculated throughPhyML using the approximate likelihood ratio test [19]A sequence from the deer (Dama dama) was used as anoutgroup

After species identification phylogenetic trees were gen-erated to establish whether barcoding information is suffi-cient to establish the geographic origin of samples A datasetwas created with all available COI sequences of the testspecies set A phylogenetic tree for this dataset was generatedas above The sequences included in this analysis are listed inSupplementary Table 2

3 Results

Of the 11 species examined the COI genes of 9 species wereamplified successfully The most representative sequence of


Aepycerotinae 1

1

085

099

099

1083

076 098

08

09

091

077078

1

1093

094096

092083

092086

079

Bovinae

Antilopinae

Alcelaphinae


Hippotraginae

Caprinae

Reduncinae

Reedbuck Peleinae

minus

DeerIMPALA

ImpalaCowZebu

BongoSitatunga

BUSHBUCKBushbuck

Greater Kudu

Lesser Kudu

NyalaELAND


BUFFALOBuffalo


SassabieWILDEBEEST

OryxAddax

SABLESable

GoatSheep

Roan Antelope

WATERBUCKWaterbuck

KAFUE LECHWEPUKU

Kafue Lechwe







N

E

K

T

ZAM

Z B

SA

T1

T10

T12T4

T9

T15

T5

T7T3

N1

T2

T2

T7

T2

T1

T1

T5T8

T6

T4T3

T9

T6

T5 T1

T4

T3

T2

T3

K14T4

T1

T2 T11E2

E4

E3

B9Z5B5Z6

E5E6

T3K12

K13

K15K16

K17

T10

T10

T1

T6 T2

T3

T4T11

T13

T14ZAM

ZAM

ZAM

Impala

Eland

Bushbuck

SA B3

B2Buffalo

SA1

ZAM

Waterbuck

ZAM

ZAM

K18E1B1

E7Z3Z2Z1T5T8T9T7K19

Z4

B8B7

B6

Z7K20 B4

Wildebeest




4 Discussion




5 Conclusion



Competing Interests


Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Aepycerotinae 1

1

085

099

099

1083

076 098

08

09

091

077078

1

1093

094096

092083

092086

079

Bovinae

Antilopinae

Alcelaphinae


Hippotraginae

Caprinae

Reduncinae

Reedbuck Peleinae

minus

DeerIMPALA

ImpalaCowZebu

BongoSitatunga

BUSHBUCKBushbuck

Greater Kudu

Lesser Kudu

NyalaELAND


BUFFALOBuffalo


SassabieWILDEBEEST

OryxAddax

SABLESable

GoatSheep

Roan Antelope

WATERBUCKWaterbuck

KAFUE LECHWEPUKU

Kafue Lechwe







N

E

K

T

ZAM

Z B

SA

T1

T10

T12T4

T9

T15

T5

T7T3

N1

T2

T2

T7

T2

T1

T1

T5T8

T6

T4T3

T9

T6

T5 T1

T4

T3

T2

T3

K14T4

T1

T2 T11E2

E4

E3

B9Z5B5Z6

E5E6

T3K12

K13

K15K16

K17

T10

T10

T1

T6 T2

T3

T4T11

T13

T14ZAM

ZAM

ZAM

Impala

Eland

Bushbuck

SA B3

B2Buffalo

SA1

ZAM

Waterbuck

ZAM

ZAM

K18E1B1

E7Z3Z2Z1T5T8T9T7K19

Z4

B8B7

B6

Z7K20 B4

Wildebeest




4 Discussion




5 Conclusion



Competing Interests


Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


N

E

K

T

ZAM

Z B

SA

T1

T10

T12T4

T9

T15

T5

T7T3

N1

T2

T2

T7

T2

T1

T1

T5T8

T6

T4T3

T9

T6

T5 T1

T4

T3

T2

T3

K14T4

T1

T2 T11E2

E4

E3

B9Z5B5Z6

E5E6

T3K12

K13

K15K16

K17

T10

T10

T1

T6 T2

T3

T4T11

T13

T14ZAM

ZAM

ZAM

Impala

Eland

Bushbuck

SA B3

B2Buffalo

SA1

ZAM

Waterbuck

ZAM

ZAM

K18E1B1

E7Z3Z2Z1T5T8T9T7K19

Z4

B8B7

B6

Z7K20 B4

Wildebeest




4 Discussion




5 Conclusion



Competing Interests


Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion




5 Conclusion



Competing Interests


Acknowledgments


References































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Cytochrome c Oxidase Sequences of Zambian ...downloads.hindawi.com/journals/ijz/2016/1808912.pdf · License, which permits unrestricted use, distribution, and reproduction