Research ArticleMitochondrial DNA Phylogenetics of Black Rhinoceros inKenya in relation to Southern Africa Population

Elijah K Githui12 David N Thuo3 Joshua O Amimo3

NyamuM Njagi2 andMaryanne M Gitari23

1 Institute of Primate Research PO Box 24481 Karen Nairobi 00502 Kenya2National Museums of Kenya PO Box 40658 Nairobi 00100 Kenya3University of Nairobi PO Box 30197 Nairobi 00100 Kenya

Correspondence should be addressed to Elijah K Githui kegithuiyahoocom

Received 11 May 2017 Revised 6 July 2017 Accepted 20 July 2017 Published 22 August 2017

Academic Editor Alexandre Sebbenn

Copyright copy 2017 Elijah K Githui et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Black rhinoceros (Diceros bicornis) are highly endangered due to poaching and other anthropological reasons and their protectionto rebound the numbers and genetic improvement are necessary remedial measures defined by Rhino International Union ofConservation for the Nature Red List (IUCN) In Kenya black rhino numbers declined from approximately 20000 in the 1970s tofewer than 400 in 1982 Wildlife conservation managers effected strategies to managebreed the remaining rhinoceros populationsin Eastern and Southern Africa within regional sanctuaries This study analyzes the genetic variability of these remnant rhinocerosusing Mitochondrial DNA (mtDNA) Majority of the rhinoceros in both Kenyan and Southern Africa group are monophyleticclusters with insignificant genetic variations while some lineages are underrepresented The Eastern Africa rhinoceros forms adistinct clade from the Sothern Africa counterpart while Tanzania population has admixtures Tajima-D test showed that these twopopulations are under different selection pressure possibly due to different history of adverse anthropologic activities Similarlythe Southern Africa rhinoceros have low genetic diversity compared to the Eastern African population due to extended periodsof game hunting during Africa colonization This study suggests that managed translocations of individual rhinoceros across theseparated fragments can be applied to improve their genetic diversity

1 Introduction

Black rhinoceros (Diceros bicornis) has suffered dramaticdecline of all mammals in the recent history and the species iscurrently categorized as critically endangered in the Interna-tional Union of Conservation for the Nature Red List (IUCN)[1 2] Between 1960s and 1980s wanton illegal poaching andloss of habitat due to increased human developments in areasthat were formerly wilderness resulted in approximately 96decline (65000 to 3800 individuals) in population blackrhinoceros across their range in Africa [3 4] In recent yearsconservation measures have resulted in increase of in situblack rhino numbers from of 2475 individuals in 1993 toapproximately 4880 in 2010 [2 5 6]

Kenya Wildlife Service (KWS) records show that theseimpacts reflected on Kenyan black rhinoceros numbers

where catastrophic decline was from an estimated 20000individuals in 1970 to 398 in 1991 and then this slightlyrebounded to about 631 in 2014 [7 8] This was throughconservation strategy developed to intensively manage theremaining rhinos within small rhino sanctuaries where KWSperiodical translocated the surviving rhinoceros into the highsecurity sanctuaries to limit poaching and enhance breeding[9 10]

Majority of the extant black rhinoceros belong to sub-species Diceros bicornis minor and Diceros bicornis michaeliOther subspecies include Diceros bicornis longipes andDiceros bicornis bicornis The D b michaeli (eastern blackrhino) is found in Kenya and Tanzania while D b minor isfound in Tanzania to South Africa [3 11] Although there areapparently no marked geographic or reproductive barriersbetween the subspecies they occupy different ecological

HindawiInternational Journal of BiodiversityVolume 2017 Article ID 8326361 6 pageshttpsdoiorg10115520178326361

2 International Journal of Biodiversity

zonesThere have not been any rigorous studies onmigrationand reproductive gene flow between the subspecies althoughsome authors suggest that each subspecies may have distinctgenetic or behavioral adaptations to their local environments[3 12]

Genetic differences between related populations can alsoresult from strong genetic drift caused by population frag-mentation and declining population sizes [13 14] Whenthe genetic structure and historic pattern of gene flow of aspecies had been described reintroduction methods can beused to secure locally adapted populations or restocking usedfor genetic supplementation Mitochondrial DNA (mtDNA)phylogeny sheds light on founder female populations andcan be applied in determining black rhinoceros populationrelationships across the ecological and geographic zones[15 16] The measure of variability within and betweengroups is an essential step in determining robust numbers ofindividuals in a sanctuary that preserves genetic diversity ofthese populations [12 17] In this study we analyzed mtDNAgenetic relationships between Kenyan black rhinoceros pop-ulation and the Southern Africa counterpart to assess geneflow between the populations

2 Materials and Methods

21 Sample Collections Samples were collected during rhinotranslocations exercises between sanctuaries within Kenyabetween years 2008 and 2012 conducted by KWS rangersand scientist at Nairobi National Park Blood and ear snipswere collected by KWS veterinary officer from anaesthetizedanimals and preserved in absolute ethanol in cryovialsinstantly Materials for laboratory analysis were transportedto Molecular Genetics Laboratory National Museums ofKenya and preserved

22 DNAExtraction Portion of skin tissue sample and bloodsample precipitates were centrifuged briefly and ethanolaspirated The samples were left to air-dry then washed in3 volumes of 1x PBS to rehydrate DNA was subsequentlyextracted using Qiagen Kit according to manufacturerrsquosmanual (Qiagen Inc Germantown MD USA) essentiallyinvolving lysis binding of DNA to silica matrix and elutionin 200 ul TE (10mM Tris HCL 1mM EDTA and pH 80)For tissues that were difficult to completely lyse phenolchloroform method was applied [18] The bloodtissue wasincubated in 50 ugml proteinase K 1 SDS in STE buffer(150mM NaCl 100mM EDTA 10mM Tris HCl and pH74) at 55∘C for 3 hrs The lysate was subjected to phenolchloroform phase separation and the upper aqueous phasecontaining DNA aspirated to a new tube DNA precipitatedfrom the aqueous phase by adding 2-3 volumes of absoluteethanol The pellet was suspended in 50 to 100 ul TE (10mMTris HCL 1mMEDTA and pH 80) DNA concentration wasmeasured by absorbance at 260 nm280 nm and the qualityanalyzed by electrophoresis in 1 Agarose gel in 1x TAEbuffer (40mM Tris acetate 1mM EDTA and pH 80) Therespective tubes with DNA were appropriately labeled andstored at minus20∘C

23 PCR and Product Purification Procedures In this study28 DNA samples representing rhinoceros from within Kenyasanctuaries and national parks were analyzed Polymerasechain reaction was carried out using the following parame-ters denaturation at 94∘C 1min annealing at 54∘C 45 secand extension at 72∘C for 1min The primers flanking regionof 31015840 end of cytochrome b and D-loop middle region wereappliedThe amplification product was verified on 1 agarosegels and the fragment excised from the gel solubilized insodium iodide solution then bound to (silica) column inthe gene clean procedure Bound DNA was eluted in 30 ulnuclease free ddH2O

24 Sequencing Gene cleaned DNA of the amplified frag-ments were sequenced at Macrogen Inc Europe (Nether-lands) using Sangerrsquos fluorescent dye chain terminatormethod Each of the samples was independently sequencedthree times and the raw sequences analyzed in Clustal-Wprogram in BioEdit (Version 705) to give the consensusbut five of the sample sequences did not yield good dataand were excluded The edited raw sequences were sub-mitted to National Center for Biotechnology Information(NCBI httpswwwncbinlmnihgov) nucleotide databaseGenBank accession numbers KU569499-KU569508 andKP247507-KP247521

25 Sequence Alignment and Phylogenetic Analysis Sequen-ces from the dataset were initially aligned utilizing theClustal-W program in BioEdit and the phylogenetic relation-ships inferred from the aligned nucleotide sequences by theNJ method (bootstrap 1000 replicates) implemented in theMEGA suite version 6 [19 20]

Test of neutrality was performed using Tajimarsquos D soft-ware implemented in MEGA suite [21 22] and the analysisof allele diversity haplotypes and population divergencedone in DNA sequence polymorphism statistics packagesimplemented in DnaSP V5 software [23 24]

3 Results

31 Distant Phylogenetic Relationship in Rhinoceros FamilyPhylogenetic tree clusters generated by NJ method at boot-strap 1000 replicates were rooted against outgroupDicerorhi-nus sumatrensis harrissoni indicating long distance evolution-ary separation between the white rhinoceros (Ceratotheriumsimum simum) and the black rhinoceros (D bicornis) clustersThe two clusters of black rhinoceros (D b minor and Db michaeli) are phylogenetically closely related Majorityof the rhinoceros in both Kenyan and Southern Africagroup are monophyletic clusters with insignificant geneticvariations while some of the representative lineages haveonly a few rhinoceros The Tanzanian rhinoceros population(Figure 1 shown in grey) is an independent lineage of Db michaeli with some admixtures from Kenya populationand possible lineage variant represented by D b rovumaevoucher sample Some rhinoceros from the South WesternAfrica (D b bicornis) cluster together with the D b minorpopulation of Southern Africa while other independentlineages from SouthWestern African population represented

International Journal of Biodiversity 3

Diceros bicornis michaeli isolate 04114 Diceros bicornis michaeli isolate LNNP-530 Diceros bicornis michaeli isolate Tatu60 Diceros bicornis michaeli isolate Peril Diceros bicornis michaeli haplotype 15 Diceros bicornis michaeli isolate Tatu603 Diceros bicornis michaeli isolate 04120 Diceros bicornis michaeli isolate Beth

Diceros bicornis michaeli isolate Latie Diceros bicornis michaeli haplotype 09

Diceros bicornis michaeli isolate Zalia Diceros bicornis michaeli isolate Bativa

AF187835 Diceros bicornis michaeli Diceros bicornis michaeli isolate Magudel Diceros bicornis michaeli haplotype 10

Diceros bicornis michaeli isolate Kiserian Diceros bicornis michaeli isolate 116 Diceros bicornis michaeli isolate Ridgeback Diceros bicornis michaeli isolate Leakey

Diceros bicornis michaeli isolate LNNP Diceros bicornis michaeli isolate Solio3

Diceros bicornis michaeli haplotype 04

Kenyan

AF187834 Diceros bicornis michaeli KY472533 Diceros bicornis michaeli voucher 8192 Tanzania

KY472390 Diceros bicornis michaeli voucher 56146 Tanzania KY472392 Diceros bicornis rovumae voucher 19481287 Tanzania

D b michaeli

KY472386 Diceros bicornis angolensis voucher MOS Angola KY472599 Diceros bicornis occidentalis voucher A247 Namibia AF187830 Diceros bicornis minor KM095539 Diceros bicornis minor voucher 1247

AF187829 Diceros bicornis minor AF187826 Diceros bicornis minor KM095529 Diceros bicornis voucher 1006 KM095570 Diceros bicornis minor voucher 1190 Diceros bicornis minor haplotype I

KY472316 Diceros bicornis bicornis voucher 19147 S Africa KY472345 Diceros bicornis minor voucher 4194NZG S Africa KM0956221 Diceros bicornis minor voucher 1100

Diceros bicornis minor haplotype II AF187832 Diceros bicornis minor AF187825 Diceros bicornis minor KM095538 Diceros bicornis minor voucher 1243

AF187833 Diceros bicornis minor KM095628 Diceros bicornis minor voucher A340 KM095530 Diceros bicornis minor voucher 1021

D b minor

Ceratotherium simum simum haplotype 4 AF187836 Ceratotherium simum simum Ceratotherium simum simum haplotype 1

Ceratotherium simum cottoni haplotype 1 Dicerorhinus sumatrensis harrissoni isolate SR04

4899

66

72

40

3488

49

57

34

47

12

22

25

43

38

60

8899

43

84

26

95

30

66

17

19

3426

24

47

71

005

Figure 1 Evolutionary relationship among global rhinoceros inferred using the NJ method The percentage of replicate trees in which theassociated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branchesThe tree is drawn to scale with branchlengths inferring evolutionary distances The analysis involved 50 nucleotide sequences and there were a total of 254 positions in the finaldataset Analyses were conducted in MEGA6

4 International Journal of Biodiversity

Table 1 Tajimarsquos neutrality test

119898 119878 119901119904 Θ 120587 119863

D b minor 16 6 0023529 0007091 0009706 1262277D b michaeli 16 10 0039216 0011818 0011667 minus0047838The analysis involved 16 nucleotide sequencesThere were a total of 255 positions (119899) in the final dataset of each populationD b minor andD b michaeli119898 =number of sequences 119899 = total number of sites 119878 = number of segregating sites 119901119904 = 119878119899Θ = 1199011199041198861 120587 = nucleotide diversity and119863 is the Tajima test statistic(120587 and 1198781198861 both estimate Θ where 119864 (expected) 119864[120587] = Θ and 119864[119878] = 1198861Θ) software default significant at 119875 lt 010 Analyses were conducted in MEGA6

Table 2 Diversity differences and genetic divergence between Black Rhinoceros population in Kenyan versus Southern Africa group(significance 119875 lt 010) conducted using DnaSP V5 software

Population 1 D b michaeli (Kenya) Population 2 D b minor (Southern Africa)Number of sequences 16 Number of sequences 16Number of polymorphic sites (haplotypes) 10 Number of polymorphic sites (haplotypes) 6Nucleotide diversity (per site) Pi 001167 Nucleotide diversity (per site) Pi 000971Haplotype (gene) diversity 0933 Haplotype diversity Hd 0900Between populations number of fixed differences 7 mutations polymorphic in population 1 but monomorphic in population 2 9 mutations polymorphic inpopulation 2 but monomorphic in population 1 5 shared mutations 1

by D b angolensis and D b occidentallis are monophyletic(Figure 1) In each black rhinoceros population tree clustersare monophyletic

32 Population Departure from Neutrality When populationis at equilibrium neutrality the nucleotide diversity (120587) andthe number of nucleotide segregating sites (Θ) are indis-tinguishable and this is seen in both Southern Africa (Db minor) and Kenyan (D b michaeli) rhinoceros Tajima-D analysis of Southern Africa rhinoceros dataset showed apositive valuewhile Kenyan group subset had aweak negativevalue (Table 1)

33 DNA Diversity and Divergence between PopulationsDNA diversity analysis of similar region of mtDNA of theKenyan and Southern African rhinoceros showed that thetwo populations are distinct with only one shared mutationand nine fixed differences The Southern Africa rhinocerospopulation had lower haplotype diversity and nucleotidediversity relative to the Kenyan population (Table 2)

4 Discussion

Black rhinoceros became critically endangered species in the1970s mainly due to poaching and historical game huntingduring colonial era and also due to expansion of agricul-tural land in Africa [25ndash27] prompting implementation ofremedial conservation strategies to intensively manage theremaining population within high security sanctuaries [59] These management fragments started with low num-bers of founding population with periodical translocationbetween the sanctuaries managed in coordination with localgovernment wild life services [7 28] The low numbers offounding population have implications in genetic variabilityand resilience of the subspecies

The Eastern Africa black rhinoceros (D b michaeli) ishistorically separated from the southern population (D bminor) although there is no real geographical barrier to limit

movement between the two regions Some authors suggestthat each subspecies population may have behavioral adapta-tions to their local environments [3 12] The two subspeciesare not genetically reproductively separated and there havebeen no rigorous studies on migration and reproductivegene flow between these populations [12] In this studyphylogenetic analysis of these populations using mtDNAD-loop region (Figure 1) shows that the two populationsfall into two different clades that have further separatedinto monophyla clusters over time This is concurrent withprevious observations that the D b michaeli and D bminor populations become distinct genetically [27 29 30]However the phylogenetic tree branch lengths of two cladesof black rhinoceros are closely linked and are distantly relatedto white rhinoceros C s simum and Sumatran rhinocerosD s harrissoni [16] indicating that the Eastern Africa andSouthern Africa black rhinoceros separation is recent TheTanzanian D b michaeli cluster has admixtures populationswith lineages from Kenya likely at the geographic areasseparated by national boundary but also has lineages close tosouthern black rhinocerosThe voucher specimen AF187834(Figure 1) was born in Cincinnati zoo Ohio USA froma Kenya lineage [31] while D b rovumae voucher is nowextinct but has closely related lineages in the southern blackrhinoceros that can be used in genetic supplementation [2731] Southwestern Africa black rhinoceros (D b bicornis)from Namibia and Angola clustered within the D b minorclade The Southwestern Africa monophyla lineages forexample D b congolensis and D b occidentalis can furtherbe studied with the aim of providing source of heterogeneityin genetic supplementation of the Southern Africa blackrhinoceros population

Inbreeding depression versus outbreeding depression areconcerns in management policies of rhinoceros populationsin sanctuaries [32 33] The current sanctuaries hold frag-mented pockets of rhinoceros after near demise in the 1970sdue to anthropologic issues but mainly poaching Previousstudies based mtDNA diversity showed that D b minor

International Journal of Biodiversity 5

population in Southern Africa had only a few haplotypesraising the question of whether these remnant populationslost genetic diversity recently as a result of managementfragments gene flow bottleneck or have been a geneticallyseparate lineage for longer time [29] Tajimarsquos D test analysisin this study showed a positive value for the southern Db minor population while the Eastern Africa D b michaelipopulation had negative value (Table 1) indicating that thetwo populations are experiencing different selection pressureBased on the recent history of demise and restocking ofAfrican rhinoceros it is plausible that the positive TajimarsquosD value reflects recent bottleneck in these fragmented pop-ulation while the Eastern African negative Tajimarsquos D valueis a result of recent population expansion from few foundingindividual rhinoceroses

For the same sample size the Eastern African D bmichaeli had higher genetic diversity (haplotypes = 10)compared to the Southern Africa D b minor (haplotypes =6) with seven fixed differences and only one shared muta-tion (Table 2) The apparent low diversity in the SouthernAfrica black rhinoceros infer a population bottleneck [2729] For the mtDNA region analyzed the fixed nucleotidepolymorphic site differences between the two populationswere seven with only one shared polymorphic site indicatingthat gene flow between the Eastern and Southern African andrhinoceros populations is restricted Since mtDNA is mater-nally inherited this may imply that the female rhinocerosfounding population has behavioral restricted movementswithin an ecological range

There is need to readjust current conservation man-agement paradigms for the black rhinoceros where precau-tion strategies in the translocation of endangered speciesbring only small incremental improvements [27 28 34]Rhinoceros are herbivores without substantial predators andare resilient to ecological challenges [35] therefore theirtranslocation for restocking to improve genetic diversity isfeasible Since there is no reproductive barrier in Africanblack rhinoceroses the genetic diversity improvement willinvolve pilot outbreeding programs among the fragmentedpopulations across the ecological range [5 27 29]

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This research project was supported by Molecular GeneticsLaboratory at National Museums of Kenya and subgrantfrom Professor Thomas Gilbert University of CopenhagenNatural History Museums Denmark to support Masters ofArts Program for Nyamu M Njagi and Masters of Sciencedissertation project for David N Thuo

References

[1] J N Garnier M W Bruford and B Goossens ldquoMating systemand reproductive skew in the black rhinocerosrdquo MolecularEcology vol 10 no 8 pp 2031ndash2041 2001

[2] R Emslie ldquoDiceros bicornisrdquo in IUCN 2011 IUCN Red List ofThreatened Species Version 20112 2011 httpwwwiucnredlistorg

[3] R Emslie and M Brooks ldquoAfrican Rhino Status Survey andConservation Action Planrdquo 1999 IUCNSSC African RhinoSpecialist Group IUCN Gland Switzerland and CambridgeUK

[4] S M Muya M W Bruford A W-T Muigai et al ldquoSubstantialmolecular variation and low genetic structure in Kenyarsquos blackrhinoceros implications for conservationrdquoConservationGenet-ics vol 12 no 6 pp 1575ndash1588 2011

[5] IUCN ldquoPosition statement on the translocation of living organ-isms introductions re-introductions and re-stockingrdquo 1987Gland Switzerland and Cambridge UK IUCN The WorldConservation Union

[6] IUCN SSC AfRSG ldquoDiceros bicornisrdquo 2008 IUCN red list ofthreatened species IUCN 2008

[7] R H Emslie andMH Knight ldquoUpdate onAfrican rhino statusand trends from IUCN SSC African Rhino Specialist Group(AfRSG)rdquo inReport to CITES Standing Committee 65thMeetingpp 1ndash6 2014

[8] D N Thuo J O Junga J M Kamau J O Amimo F MKibegwa and K E Githui ldquoPopulation viability analysis ofblack rhinoceros (Diceros bicornis michaeli) in Lake NakuruNational Park Kenyardquo Journal of Biodiversity amp EndangeredSpecies vol 3 article 1 2015

[9] R A Brett ldquoConservation Strategy and Management Plan forthe Black Rhinoceros (Diceros bicornis) in Kenyardquo in RhinoConservation Programme Kenya Wildlife Service 1993

[10] R H Emslie R Amin and R Kock ldquoGuidelines for the insitu re-introduction and translocation of African and Asianrhinocerosrdquo 2009 Occasional paper of the IUCN SpeciesSurvival Commission No 39

[11] C P Groves ldquoGeographic variation in the black rhinocerosDiceros bicornis (L 1758)rdquo Zeitschrift fur Saugetierkunde vol32 pp 267ndash276 1967

[12] E H Harley I Baumgarten J Cunningham and C OrsquoRyanldquoGenetic variation andpopulation structure in remnant popula-tions of black rhinoceros Diceros bicornis inAfricardquoMolecularEcology vol 14 no 10 pp 2981ndash2990 2005

[13] R Frankham J D Ballou M D B Eldridge et al ldquoPredictingthe probability of outbreeding depressionrdquo Conservation Biol-ogy vol 25 no 3 pp 465ndash475 2011

[14] F W Allendorf and G Luikart Conservation and the Geneticsof Populations Blackwell Malden Mass USA 2007

[15] M Jama Y Zhang R A Aman and O A Ryder ldquoSequenceof the mitochondrial control region tRNA119905ℎ119903 tRNS119901119903119900 andtRNA119901ℎ119890 genes from the black rhinoceros Diceros bicornisrdquoNucleic Acids Research vol 21 no 18 article 4392 1993

[16] E Willerslev M T P Gilbert J Binladen et al ldquoAnalysisof complete mitochondrial genomes from extinct and extantrhinoceroses reveals lack of phylogenetic resolutionrdquo BMCEvolutionary Biology vol 9 article 95 2009

[17] J C Avise J E Neigel and J Arnold ldquoDemographic influenceson mitochondrial DNA lineage survivorship in animal popula-tionsrdquo Journal of Molecular Evolution vol 20 no 2 pp 99ndash1051984

[18] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbour Laboratory PressNew York NY USA 1989

4 International Journal of Biodiversity

Table 1 Tajimarsquos neutrality test

119898 119878 119901119904 Θ 120587 119863

D b minor 16 6 0023529 0007091 0009706 1262277D b michaeli 16 10 0039216 0011818 0011667 minus0047838The analysis involved 16 nucleotide sequencesThere were a total of 255 positions (119899) in the final dataset of each populationD b minor andD b michaeli119898 =number of sequences 119899 = total number of sites 119878 = number of segregating sites 119901119904 = 119878119899Θ = 1199011199041198861 120587 = nucleotide diversity and119863 is the Tajima test statistic(120587 and 1198781198861 both estimate Θ where 119864 (expected) 119864[120587] = Θ and 119864[119878] = 1198861Θ) software default significant at 119875 lt 010 Analyses were conducted in MEGA6

Table 2 Diversity differences and genetic divergence between Black Rhinoceros population in Kenyan versus Southern Africa group(significance 119875 lt 010) conducted using DnaSP V5 software

Population 1 D b michaeli (Kenya) Population 2 D b minor (Southern Africa)Number of sequences 16 Number of sequences 16Number of polymorphic sites (haplotypes) 10 Number of polymorphic sites (haplotypes) 6Nucleotide diversity (per site) Pi 001167 Nucleotide diversity (per site) Pi 000971Haplotype (gene) diversity 0933 Haplotype diversity Hd 0900Between populations number of fixed differences 7 mutations polymorphic in population 1 but monomorphic in population 2 9 mutations polymorphic inpopulation 2 but monomorphic in population 1 5 shared mutations 1

by D b angolensis and D b occidentallis are monophyletic(Figure 1) In each black rhinoceros population tree clustersare monophyletic

32 Population Departure from Neutrality When populationis at equilibrium neutrality the nucleotide diversity (120587) andthe number of nucleotide segregating sites (Θ) are indis-tinguishable and this is seen in both Southern Africa (Db minor) and Kenyan (D b michaeli) rhinoceros Tajima-D analysis of Southern Africa rhinoceros dataset showed apositive valuewhile Kenyan group subset had aweak negativevalue (Table 1)

33 DNA Diversity and Divergence between PopulationsDNA diversity analysis of similar region of mtDNA of theKenyan and Southern African rhinoceros showed that thetwo populations are distinct with only one shared mutationand nine fixed differences The Southern Africa rhinocerospopulation had lower haplotype diversity and nucleotidediversity relative to the Kenyan population (Table 2)

4 Discussion

Black rhinoceros became critically endangered species in the1970s mainly due to poaching and historical game huntingduring colonial era and also due to expansion of agricul-tural land in Africa [25ndash27] prompting implementation ofremedial conservation strategies to intensively manage theremaining population within high security sanctuaries [59] These management fragments started with low num-bers of founding population with periodical translocationbetween the sanctuaries managed in coordination with localgovernment wild life services [7 28] The low numbers offounding population have implications in genetic variabilityand resilience of the subspecies

The Eastern Africa black rhinoceros (D b michaeli) ishistorically separated from the southern population (D bminor) although there is no real geographical barrier to limit

movement between the two regions Some authors suggestthat each subspecies population may have behavioral adapta-tions to their local environments [3 12] The two subspeciesare not genetically reproductively separated and there havebeen no rigorous studies on migration and reproductivegene flow between these populations [12] In this studyphylogenetic analysis of these populations using mtDNAD-loop region (Figure 1) shows that the two populationsfall into two different clades that have further separatedinto monophyla clusters over time This is concurrent withprevious observations that the D b michaeli and D bminor populations become distinct genetically [27 29 30]However the phylogenetic tree branch lengths of two cladesof black rhinoceros are closely linked and are distantly relatedto white rhinoceros C s simum and Sumatran rhinocerosD s harrissoni [16] indicating that the Eastern Africa andSouthern Africa black rhinoceros separation is recent TheTanzanian D b michaeli cluster has admixtures populationswith lineages from Kenya likely at the geographic areasseparated by national boundary but also has lineages close tosouthern black rhinocerosThe voucher specimen AF187834(Figure 1) was born in Cincinnati zoo Ohio USA froma Kenya lineage [31] while D b rovumae voucher is nowextinct but has closely related lineages in the southern blackrhinoceros that can be used in genetic supplementation [2731] Southwestern Africa black rhinoceros (D b bicornis)from Namibia and Angola clustered within the D b minorclade The Southwestern Africa monophyla lineages forexample D b congolensis and D b occidentalis can furtherbe studied with the aim of providing source of heterogeneityin genetic supplementation of the Southern Africa blackrhinoceros population

Inbreeding depression versus outbreeding depression areconcerns in management policies of rhinoceros populationsin sanctuaries [32 33] The current sanctuaries hold frag-mented pockets of rhinoceros after near demise in the 1970sdue to anthropologic issues but mainly poaching Previousstudies based mtDNA diversity showed that D b minor

International Journal of Biodiversity 5

population in Southern Africa had only a few haplotypesraising the question of whether these remnant populationslost genetic diversity recently as a result of managementfragments gene flow bottleneck or have been a geneticallyseparate lineage for longer time [29] Tajimarsquos D test analysisin this study showed a positive value for the southern Db minor population while the Eastern Africa D b michaelipopulation had negative value (Table 1) indicating that thetwo populations are experiencing different selection pressureBased on the recent history of demise and restocking ofAfrican rhinoceros it is plausible that the positive TajimarsquosD value reflects recent bottleneck in these fragmented pop-ulation while the Eastern African negative Tajimarsquos D valueis a result of recent population expansion from few foundingindividual rhinoceroses

For the same sample size the Eastern African D bmichaeli had higher genetic diversity (haplotypes = 10)compared to the Southern Africa D b minor (haplotypes =6) with seven fixed differences and only one shared muta-tion (Table 2) The apparent low diversity in the SouthernAfrica black rhinoceros infer a population bottleneck [2729] For the mtDNA region analyzed the fixed nucleotidepolymorphic site differences between the two populationswere seven with only one shared polymorphic site indicatingthat gene flow between the Eastern and Southern African andrhinoceros populations is restricted Since mtDNA is mater-nally inherited this may imply that the female rhinocerosfounding population has behavioral restricted movementswithin an ecological range

There is need to readjust current conservation man-agement paradigms for the black rhinoceros where precau-tion strategies in the translocation of endangered speciesbring only small incremental improvements [27 28 34]Rhinoceros are herbivores without substantial predators andare resilient to ecological challenges [35] therefore theirtranslocation for restocking to improve genetic diversity isfeasible Since there is no reproductive barrier in Africanblack rhinoceroses the genetic diversity improvement willinvolve pilot outbreeding programs among the fragmentedpopulations across the ecological range [5 27 29]

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This research project was supported by Molecular GeneticsLaboratory at National Museums of Kenya and subgrantfrom Professor Thomas Gilbert University of CopenhagenNatural History Museums Denmark to support Masters ofArts Program for Nyamu M Njagi and Masters of Sciencedissertation project for David N Thuo

References

[1] J N Garnier M W Bruford and B Goossens ldquoMating systemand reproductive skew in the black rhinocerosrdquo MolecularEcology vol 10 no 8 pp 2031ndash2041 2001

[2] R Emslie ldquoDiceros bicornisrdquo in IUCN 2011 IUCN Red List ofThreatened Species Version 20112 2011 httpwwwiucnredlistorg

[3] R Emslie and M Brooks ldquoAfrican Rhino Status Survey andConservation Action Planrdquo 1999 IUCNSSC African RhinoSpecialist Group IUCN Gland Switzerland and CambridgeUK

[4] S M Muya M W Bruford A W-T Muigai et al ldquoSubstantialmolecular variation and low genetic structure in Kenyarsquos blackrhinoceros implications for conservationrdquoConservationGenet-ics vol 12 no 6 pp 1575ndash1588 2011

[5] IUCN ldquoPosition statement on the translocation of living organ-isms introductions re-introductions and re-stockingrdquo 1987Gland Switzerland and Cambridge UK IUCN The WorldConservation Union

[6] IUCN SSC AfRSG ldquoDiceros bicornisrdquo 2008 IUCN red list ofthreatened species IUCN 2008

[7] R H Emslie andMH Knight ldquoUpdate onAfrican rhino statusand trends from IUCN SSC African Rhino Specialist Group(AfRSG)rdquo inReport to CITES Standing Committee 65thMeetingpp 1ndash6 2014

[8] D N Thuo J O Junga J M Kamau J O Amimo F MKibegwa and K E Githui ldquoPopulation viability analysis ofblack rhinoceros (Diceros bicornis michaeli) in Lake NakuruNational Park Kenyardquo Journal of Biodiversity amp EndangeredSpecies vol 3 article 1 2015

[9] R A Brett ldquoConservation Strategy and Management Plan forthe Black Rhinoceros (Diceros bicornis) in Kenyardquo in RhinoConservation Programme Kenya Wildlife Service 1993

[10] R H Emslie R Amin and R Kock ldquoGuidelines for the insitu re-introduction and translocation of African and Asianrhinocerosrdquo 2009 Occasional paper of the IUCN SpeciesSurvival Commission No 39

[11] C P Groves ldquoGeographic variation in the black rhinocerosDiceros bicornis (L 1758)rdquo Zeitschrift fur Saugetierkunde vol32 pp 267ndash276 1967

[12] E H Harley I Baumgarten J Cunningham and C OrsquoRyanldquoGenetic variation andpopulation structure in remnant popula-tions of black rhinoceros Diceros bicornis inAfricardquoMolecularEcology vol 14 no 10 pp 2981ndash2990 2005

[13] R Frankham J D Ballou M D B Eldridge et al ldquoPredictingthe probability of outbreeding depressionrdquo Conservation Biol-ogy vol 25 no 3 pp 465ndash475 2011

[14] F W Allendorf and G Luikart Conservation and the Geneticsof Populations Blackwell Malden Mass USA 2007

[15] M Jama Y Zhang R A Aman and O A Ryder ldquoSequenceof the mitochondrial control region tRNA119905ℎ119903 tRNS119901119903119900 andtRNA119901ℎ119890 genes from the black rhinoceros Diceros bicornisrdquoNucleic Acids Research vol 21 no 18 article 4392 1993

[16] E Willerslev M T P Gilbert J Binladen et al ldquoAnalysisof complete mitochondrial genomes from extinct and extantrhinoceroses reveals lack of phylogenetic resolutionrdquo BMCEvolutionary Biology vol 9 article 95 2009

[17] J C Avise J E Neigel and J Arnold ldquoDemographic influenceson mitochondrial DNA lineage survivorship in animal popula-tionsrdquo Journal of Molecular Evolution vol 20 no 2 pp 99ndash1051984

[18] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbour Laboratory PressNew York NY USA 1989

International Journal of Biodiversity 5

population in Southern Africa had only a few haplotypesraising the question of whether these remnant populationslost genetic diversity recently as a result of managementfragments gene flow bottleneck or have been a geneticallyseparate lineage for longer time [29] Tajimarsquos D test analysisin this study showed a positive value for the southern Db minor population while the Eastern Africa D b michaelipopulation had negative value (Table 1) indicating that thetwo populations are experiencing different selection pressureBased on the recent history of demise and restocking ofAfrican rhinoceros it is plausible that the positive TajimarsquosD value reflects recent bottleneck in these fragmented pop-ulation while the Eastern African negative Tajimarsquos D valueis a result of recent population expansion from few foundingindividual rhinoceroses

For the same sample size the Eastern African D bmichaeli had higher genetic diversity (haplotypes = 10)compared to the Southern Africa D b minor (haplotypes =6) with seven fixed differences and only one shared muta-tion (Table 2) The apparent low diversity in the SouthernAfrica black rhinoceros infer a population bottleneck [2729] For the mtDNA region analyzed the fixed nucleotidepolymorphic site differences between the two populationswere seven with only one shared polymorphic site indicatingthat gene flow between the Eastern and Southern African andrhinoceros populations is restricted Since mtDNA is mater-nally inherited this may imply that the female rhinocerosfounding population has behavioral restricted movementswithin an ecological range

There is need to readjust current conservation man-agement paradigms for the black rhinoceros where precau-tion strategies in the translocation of endangered speciesbring only small incremental improvements [27 28 34]Rhinoceros are herbivores without substantial predators andare resilient to ecological challenges [35] therefore theirtranslocation for restocking to improve genetic diversity isfeasible Since there is no reproductive barrier in Africanblack rhinoceroses the genetic diversity improvement willinvolve pilot outbreeding programs among the fragmentedpopulations across the ecological range [5 27 29]

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This research project was supported by Molecular GeneticsLaboratory at National Museums of Kenya and subgrantfrom Professor Thomas Gilbert University of CopenhagenNatural History Museums Denmark to support Masters ofArts Program for Nyamu M Njagi and Masters of Sciencedissertation project for David N Thuo

References

[1] J N Garnier M W Bruford and B Goossens ldquoMating systemand reproductive skew in the black rhinocerosrdquo MolecularEcology vol 10 no 8 pp 2031ndash2041 2001

[2] R Emslie ldquoDiceros bicornisrdquo in IUCN 2011 IUCN Red List ofThreatened Species Version 20112 2011 httpwwwiucnredlistorg

[3] R Emslie and M Brooks ldquoAfrican Rhino Status Survey andConservation Action Planrdquo 1999 IUCNSSC African RhinoSpecialist Group IUCN Gland Switzerland and CambridgeUK

[4] S M Muya M W Bruford A W-T Muigai et al ldquoSubstantialmolecular variation and low genetic structure in Kenyarsquos blackrhinoceros implications for conservationrdquoConservationGenet-ics vol 12 no 6 pp 1575ndash1588 2011

[5] IUCN ldquoPosition statement on the translocation of living organ-isms introductions re-introductions and re-stockingrdquo 1987Gland Switzerland and Cambridge UK IUCN The WorldConservation Union

[6] IUCN SSC AfRSG ldquoDiceros bicornisrdquo 2008 IUCN red list ofthreatened species IUCN 2008

[7] R H Emslie andMH Knight ldquoUpdate onAfrican rhino statusand trends from IUCN SSC African Rhino Specialist Group(AfRSG)rdquo inReport to CITES Standing Committee 65thMeetingpp 1ndash6 2014

[8] D N Thuo J O Junga J M Kamau J O Amimo F MKibegwa and K E Githui ldquoPopulation viability analysis ofblack rhinoceros (Diceros bicornis michaeli) in Lake NakuruNational Park Kenyardquo Journal of Biodiversity amp EndangeredSpecies vol 3 article 1 2015

[9] R A Brett ldquoConservation Strategy and Management Plan forthe Black Rhinoceros (Diceros bicornis) in Kenyardquo in RhinoConservation Programme Kenya Wildlife Service 1993

[10] R H Emslie R Amin and R Kock ldquoGuidelines for the insitu re-introduction and translocation of African and Asianrhinocerosrdquo 2009 Occasional paper of the IUCN SpeciesSurvival Commission No 39

[11] C P Groves ldquoGeographic variation in the black rhinocerosDiceros bicornis (L 1758)rdquo Zeitschrift fur Saugetierkunde vol32 pp 267ndash276 1967

[12] E H Harley I Baumgarten J Cunningham and C OrsquoRyanldquoGenetic variation andpopulation structure in remnant popula-tions of black rhinoceros Diceros bicornis inAfricardquoMolecularEcology vol 14 no 10 pp 2981ndash2990 2005

[13] R Frankham J D Ballou M D B Eldridge et al ldquoPredictingthe probability of outbreeding depressionrdquo Conservation Biol-ogy vol 25 no 3 pp 465ndash475 2011

[14] F W Allendorf and G Luikart Conservation and the Geneticsof Populations Blackwell Malden Mass USA 2007

[15] M Jama Y Zhang R A Aman and O A Ryder ldquoSequenceof the mitochondrial control region tRNA119905ℎ119903 tRNS119901119903119900 andtRNA119901ℎ119890 genes from the black rhinoceros Diceros bicornisrdquoNucleic Acids Research vol 21 no 18 article 4392 1993

[16] E Willerslev M T P Gilbert J Binladen et al ldquoAnalysisof complete mitochondrial genomes from extinct and extantrhinoceroses reveals lack of phylogenetic resolutionrdquo BMCEvolutionary Biology vol 9 article 95 2009

[17] J C Avise J E Neigel and J Arnold ldquoDemographic influenceson mitochondrial DNA lineage survivorship in animal popula-tionsrdquo Journal of Molecular Evolution vol 20 no 2 pp 99ndash1051984

[18] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbour Laboratory PressNew York NY USA 1989

6 International Journal of Biodiversity

[19] K Tamura M Nei and S Kumar ldquoProspects for inferringvery large phylogenies by using the neighbor-joining methodrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 30 pp 11030ndash11035 2004

[20] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[21] F Tajima ldquoStatistical method for testing the neutral mutationhypothesis by DNApolymorphismrdquoGenetics vol 123 no 3 pp585ndash595 1989

[22] M Nei and S Kumar Molecular Evolution and PhylogeneticsOxford University Press New York NY USA 2000

[23] J Rozas J C Sanchez-DelBarrio X Messeguer and R RozasldquoDNAsp DNA polymorphism analyses by the coalescent andother methodsrdquo Bioinformatics vol 19 no 18 pp 2496-24972003

[24] P Librado and J Rozas ldquoDnaSP v5 a software for comprehen-sive analysis of DNA polymorphism datardquo Bioinformatics vol25 no 11 pp 1451-1452 2009

[25] E I Steinhart ldquoHunters poachers and gamekeepers Towardsa social history of hunting in colonial Kenyardquo The Journal ofAfrican History vol 30 no 2 pp 247ndash264 1989

[26] A Thompsell Hunting Africa British Sport African KnowledgeandTheNature of Empire (Britain andTheWorld) IntroductionReconsidering Hunting as A Site of Masculine and ImperialDomination Palgrave Macmillan London UK 2015

[27] YMoodley IM Russo D L Dalton et al ldquoExtinctions geneticerosion and conservation options for the black rhinoceros(Diceros bicornis)rdquo Scientific Reports vol 7 Article ID 414172017

[28] W L Linklater K Adcock P du Preez et al ldquoGuidelines forlarge herbivore translocation simplified black rhinoceros casestudyrdquo Journal of Applied Ecology vol 48 no 2 pp 493ndash5022011

[29] R M Anderson-Lederer W L Linklater and P A RitchieldquoLimited mitochondrial DNA variation within South Africarsquosblack rhino (Diceros bicornis minor) population and implica-tions for managementrdquo African Journal of Ecology vol 50 no4 pp 404ndash413 2012

[30] R Du Toit ldquoAfrican rhino systematicsmdashthe existing basis forsubspecies classification of black and white rhinosrdquo Pachydermvol 9 pp 3ndash7 1987

[31] S M Brown and B A Houlden ldquoConservation genetics ofthe black rhinoceros (Diceros bicornis)rdquoConservation Geneticsvol 1 no 4 pp 365ndash370 2000

[32] L C Rookmaaker ldquoReview of the European perception of theAfrican rhinocerosrdquo Journal of Zoology vol 265 no 4 pp 365ndash376 2005

[33] G Clinning DDruce D Robertson J Bird and BNxeleBlackRhino in Hluhluwe iMfolozi Park Historical Records Status ofCurrent Population and Monitoring and Future ManagementRecommendations Ezemvelo KZN Wildlife Hluhluwe SouthAfrica 2009

[34] W L Linklater J V Gedir P R Law et al ldquoTranslocationsas experiments in the ecological resilience of anasocial mega-herbivorerdquo PLoS ONE vol 7 p e30664 2012

[35] J M Scott D D Goble A M Haines J A Wiens andM C Neel ldquoConservation-reliant species and the future ofconservationrdquoConservation Letters vol 3 no 2 pp 91ndash97 2010

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology