Introduction

The Bathyergidae (African mole-rats) are a unique groupof fossorial rodents. Species range in sociality from solitarythrough social/colonial to two eusocial species, theDamaraland mole-rat Cryptomys damarensis and the nakedmole-rat Heterocephalus glaber (Jarvis 1981; Jarvis & Bennett1994). In these latter two species, groups of co-operatively

breeding individuals exhibit a reproductive division oflabour, co-operative care of offspring and overlappinggenerations, defined by Wilson (1971) as the criteria foreusociality. In both captive and wild naked mole-ratcolonies, a behavioural division of labour is also evident,the smaller animals performing higher frequencies of‘worker’ type behaviours, while larger individuals adopt adefensive role. The reproductive division of labour is man-ifest as a monopoly of breeding by a dominant female, the‘queen’, and one to three males (Jarvis 1981; Lacey &Sherman 1991). The remaining colony members of both

Micro- and macrogeographical genetic structure ofcolonies of naked mole-rats Heterocephalus glaber

C . G . F A U L K E S , D . H . A B B O T T , * H . P . O ’ B R I E N , L . L A U , † M . R . R O Y , ‡ R . K . W A Y N E † andM . W . B R U F O R DConservation Genetics Group, Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK,

*Department of OB/GYN and Wisconsin Regional Primate Research Centre, 1223 Capitol Court, Madison, Wisconsin 53713-1299,USA, †Department of Biology, University of California, Los Angeles, CA 90024, USA, ‡Zoological Institute, University ofCopenhagen, Department of Population Biology, Universitetsparken 15, DK-2100, Denmark

Abstract

Patterns of genetic structure in eusocial naked mole-rat populations were quantifiedwithin and among geographically distant populations using multilocus DNA finger-printing and mitochondrial DNA (mtDNA) sequence analysis. Individuals withincolonies were genetically almost monomorphic, sharing the same mtDNA control regionhaplotype and having coefficients of band sharing estimated from DNA fingerprintsranging from 0.93 to 0.99. Family analysis of a hybrid captive colony of naked mole-ratswith increased levels of genetic variability using multilocus DNA fingerprinting gaveresults consistent with Mendelian inheritance, and has revealed for the first time thatmultiple paternity can occur. In a survey of wild colonies from Ethiopia, Somalia andlocations in northern and southern Kenya, we have examined mtDNA control regionsequence variation in 42 individuals from 15 colonies, and together with multilocus DNAfingerprinting and mtDNA cytochrome-b sequence analysis in selected individuals haveshown that these populations show considerable genetic divergence. Most of the variancein sequence divergence was found to be between geographical locations (Φct = 0.68) andthere was a significant correlation between sequence divergence and geographical sepa-ration of haplotypes. Six colonies from Mtito Andei in southern Kenya shared the samecontrol region haplotype, suggesting a recent common maternal ancestor. In contrast, outof four colonies at Lerata in north Kenya, three haplotypes were identified, and phyloge-netic analysis suggests that this area may be a zone where two distinct lineages are inclose proximity. Genetic distances were maximal between Ethiopian and southernKenyan populations at 5.8% for cytochrome-b, and are approaching interspecific valuesseen between other Bathyergids.

Keywords: DNA fingerprinting, naked mole-rats, control region sequence, cytochrome-bsequence, genetic variability, multimale paternity

Received 30 October 1996; revision accepted 31 January 1997

Molecular Ecology 1997, 6, 615–628

© 1997 Blackwell Science Ltd

Correspondence: C. G. Faulkes. Fax: 0171-586-2870; E-mail:c.faulkes@ucl.ac.uk

sexes are reproductively quiescent, but not sterile, and areclassified as nonbreeders (Jarvis 1981; Lacey & Sherman1991; Faulkes et al. 1990a, 1991). During oestrus, the breed-ing queen appears to initiate courtship and solicits matingfrom only the breeding male. If more than one breedingmale is present, the queen may mate with the other(s) onnumerous occasions during the oestrus period (Jarvis 1991),thus presenting the opportunity for multimale paternityand the formation of more genetically heterogeneous litters.

Naked mole-rats are found in the semiarid regions ofEast Africa including parts of Kenya, Ethiopia andSomalia (Fig. 1; Honeycutt et al. 1991a). Their subterraneancolonies exceed all other bathyergid species in their groupsizes, which commonly contain 40–90, but sometimes over295 individuals, and total tunnel length within the burrowmay exceed 3 km (Brett 1991). Due to the xenophobicnature of naked mole-rats (Lacey & Sherman 1991), andthe high cost of dispersal for individuals or small groupsof animals (Lovegrove & Wissel 1988), these wild coloniesare almost completely isolated breeding groups. Newcolonies are thought to form almost exclusively throughfission of existing colonies (Brett 1991). The combination ofenvironmental constraints that limit dispersal, and thereproductive strategy of the naked mole-rat, would be

expected to produce colonies that naturally become highlyinbred, and previous genetic studies have confirmed thishypothesis (Faulkes et al. 1990b; Reeve et al. 1990; Honeycuttet al. 1991b). Reeve et al. (1990) calculated from multilocusDNA fingerprints that the average intracolony relatednesswas 0.81, and the coefficient of inbreeding (F) was 0.45, thehighest recorded for a natural mammalian population. Suchclose genetic relatedness between colony members wouldoffset the individual reproductive sacrifice made by thenonbreeders, most of whom never breed, because theirinclusive fitness is increased (Hamilton 1964).

While the occurrence of outbreeding events in wildcolonies of naked mole-rats is rare due to the high risksinvolved in dispersal (including predation, energetic costof burrowing and the xenophobic nature of colonies), lim-ited outbreeding does appear to occur (Sherman et al.1992). The recent discovery of a male outbreeding pheno-type in some captive colonies of naked mole-rats (O’Riainet al. 1996) means that, in addition to the breeders, oppor-tunities do exist for a small number of animals to increasetheir individual fitness. These are almost exclusively maleswho do not attempt to mate whilst within their natalcolonies. In the wild, such dispersal events could alsoincrease the genetic heterogeneity of colonies into which

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

616 C . G . F A U L K E S E T A L .

Fig. 1 Sampling maps showing the species range (within the dotted line), and the relative locations of catching sites in (a) Ethiopia(Dembalawachu), north Kenya (Lerata) and south Kenya (Mtito Andei), and (b) details of the distribution of colonies studied near MtitoAndei, and (c) distribution of colonies studied at Lerata.

outbreeders migrate, potentially offsetting some of thedeleterious effects of prolonged inbreeding. However,depending on the genetic composition of neighbouringcolonies within a region, there could also be a fitness costto nonbreeders within the colony into which a dispersermigrates. For example, a male disperser that is geneticallydifferent or less related to the new colony into which hemigrates and reproduces, would produce offspring thatare in turn less related to the existing nonbreeding helperswithin that colony, than those that a consanguineous mat-ing would have produced. The effect of this reduction inrelatedness within the colony would be to reduce theinclusive fitness benefits gained by the nonbreedinghelpers. Clearly, in order to understand the relative con-tributions of inbreeding, outbreeding and individual vs.inclusive fitness in the social system of the naked mole-rat,a knowledge of the genetic structure within and betweencolonies in the wild is necessary.

While earlier studies have examined allozyme variationand mtDNA restriction fragment length polymorphisms(RFLPs) within and between colonies in northern andsouthern Kenya (Honeycutt et al. 1991b), comparisonshave not been made at the potentially more informativeminisatellite loci, and direct sequencing to determine thehaplotype diversity between populations, especially out-side Kenya, has previously not been undertaken. As aresult of both the tendency to avoid outbreeding and thesubterranean niche of the naked mole-rat, gene flow mustbe limited, and one would expect geographically distantpopulations to be highly divergent. This is supported bythe limited data available, where Honeycutt et al. (1991b)used RFLP data to calculate an average nucleotidesequence divergence (δ) of 5.4% between Samburu in thenorth of Kenya, and Mtito Andei in the south.

In this study, multilocus minisatellite DNA finger-printing was used to perform a pedigree and segregationanalysis of captive families of naked mole-rats in hybridcolonies that contain increased levels of genetic variation,to look for evidence of multimale paternity. This has notpreviously been possible due to the low heterozygositynormally found in naked mole-rat colonies (Faulkes et al.1990b; Reeve et al. 1990). In addition, both DNA finger-printing and mtDNA sequence analysis were used toinvestigate genetic structure and phylogenetic history ofnaked mole-rat colonies at a micro- and macrogeo-graphical level, over a wide proportion of their rangeincluding individuals from Ethiopia and Somalia.

Materials and methods

Samples

Naked mole-rats were obtained from seven locations asfollows: (i) ‘Mtito Andei’, from five colonies located within

a 3-km radius near Tsavo National Park, Kenya, in an agri-cultural area close to the village of Kathekani, ≈ 5 km northof Mtito Andei (3°S, 38°E); (ii) ‘Ithumba’, from one colonyat Ithumba, 60 km north-east of Mtito Andei; (iii) ‘Athi’,from one colony at Athi, 30 km south-east of Mtito Andei;(iv) ‘Lerata’, from four neighbouring colonies spaced≈ 400 m apart in the Lerata region of north Kenya (1°N,38°E); (v) ‘Meru’, from one colony in the Meru region ofnorth Kenya (0°N, 38°E); (vi) ‘Ethiopia’, from one colonyin Dembalawachu, Ethiopia (4°N, 38°E) and (vii)‘Somalia’, from one colony at Rungno, near El Bur inSomalia (5°N, 46°E, Meester 1960). The relative locationsof the colonies and geographical areas are illustrated inFig. 1.

A number of different tissues were collected from theMtito Andei animals immediately post mortem and frozenin liquid nitrogen, then transferred to a freezer at – 20 °C.Skin was collected from the Ethiopian animals immed-iately post mortem and preserved in a solution of 25%dimethyl sulphoxide in saturated NaCl, then transferredto a freezer at – 20 °C. The animals caught in Lerata weremaintained in captivity at the University of Cape Town,and following death the whole body immediately frozenat – 20 °C. DNA was subsequently extracted from samplesof skin. Samples from Meru, Somalia and Athi andIthumba were taken from formalin-fixed specimensobtained from the Zoological Society of Philadelphia,USA, the Transvaal Museum, Pretoria, South Africa andDepartment of Zoology in Cape Town, respectively.Previous genetic studies of naked mole-rats (Reeve et al.1990; Faulkes et al. 1990b) have revealed a lack of geneticvariation within colonies in the wild, so to facilitate a pedigree analysis samples from two ‘hybrid’ captivecolonies were collected. Colony 2200 was composed of abreeding queen, originally a nonbreeder from a colonyarising from the pairing of a male from Mtito Andei,southern Kenya and a female from Lerata, northernKenya, a breeding male from a colony from Mtito Andeiand their offspring. DNA samples from the queen andbreeding male were extracted from skin biopsies taken fromthe tail tip, and skin taken from 13 stillborn pups from onelitter of 16 individuals. Colony N constituted the F2 gener-ation from the pairing of a male from Tsavo, southernKenya and a female from Lerata, northern Kenya. DNAsamples from the breeding queen and three putativebreeding males were extracted from skin biopsies takenfrom the tail tip, and skin taken from stillborn pups fromtwo litters of five individuals each. In both colonies thepups died neonatally of natural causes, and the tissue (tailbiopsy or whole pup) was frozen at – 20 °C immediatelyfollowing collection. Because multilocus fingerprintingrequires relatively large amounts of high-molecular-weight DNA, it was not possible to utilize the samplesfrom all geographical locations for this technique.

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 617

DNA preparation

DNA was extracted from the tissue samples using a standard protocol by homogenizing with a lysis bufferand subsequent extraction with phenol : chloroform aspreviously described by Faulkes et al. (1990b) and Brufordet al. (1992). DNA was extracted from formalin-fixedsamples using the same protocol as above, but having firstwashed the finely chopped tissue three times by vortexmixing with 5–10 volumes of distilled water, and removalof the supernatent following centrifugation to pellet thetissue.

Multilocus minisatellite DNA fingerprinting

Samples containing 6 µg of DNA were digested with therestriction endonuclease HinfI. After digestion the sampleswere purified by extracting once with phenol : chloroform,and re-precipitating the DNA with absolute alcohol. Afterdrying, the digested DNA samples were dissolved in22 µL of dH2O, and 2 µL removed and electrophoresed tocheck the quality and quantity of the digests (Bruford et al.1992). The digested DNA samples were then subjected toelectrophoresis through a 22-cm 0.8% agarose gel, for≈ 40 h at 50 V. The DNA was transferred from the gel toHybond-N nylon filters using a vacuum blotter. Mini-satellite probes (either 33.6 or 33.15; Jeffreys et al. 1985a,b)were radiolabelled by the random prime oligolabellingmethod, and filters were hybridized overnight at 62 °C.Following hybridization, filters were washed at 62 °C withsolutions of (i) 0.25 M phosphate/1% SDS;. (ii) 2XSSC/0.1% SDS, and (iii) 1X SSC/0.1% SDS, then auto-radiographed for 2–5 days at – 80 °C (Bruford et al. 1992).

For family analysis, two captive colonies were analysed(see under ‘samples’) as follows: colony 2200, queen,breeding male and 13 offspring from one litter; colony N,queen, two breeding males and five offspring each fromtwo litters. For comparisons within and between geo-graphical areas, individuals from the following colonieswere analysed: Ethiopia, colony 1 (n = 4); Lerata, northKenya, colony Lerata 4 (n = 1), colony Lerata 2 (n = 2);Mtito Andei, south Kenya, colony K (n = 5), colony 1(n = 5), colony 4 (n = 3), colony 5 (n = 1). Because variationwithin colonies was low (see results), a single individualcan be representative of the whole colony.

Analysis of DNA fingerprints

For each fingerprint, only prominent, clearly distinctbands were scored, and based on the assumption thatshared bands represent identical minisatellite alleles of thesame locus, intra and intercolony coefficients of band sharing (x) were calculated by pairwise comparison of

individual banding patterns within a gel (Bruford et al.1992).

For pedigree and segregation analysis, we used themethod described in Bruford & Burke (1994). Bandsshared between the parents or those not clearly visiblewere eliminated from the analysis. Transmission of eachparental band into the offspring was scored as ‘1’ if the band was present and ‘0’ if the band was absent in the offspring. The pattern of segregation was thenanalysed using a simple computer program. TheKolmogorov–Smirnov goodness of fit statistic D was usedto quantify the difference between the observed distribu-tion of cosegregation and the binomial distribution whichwould be expected in the case of completely random seg-regation. Computer simulations were used to generatedistributions of the values of D that would be expected fordifferent levels of recombination (ø = 0.50, 0.45, 0.40, 0.35,0.30) given the data set we obtained. One-hundred valueswere generated for each value of ø for colony 2200 (ninematernal loci, three paternal loci and 13 offspring) andcolony N (five maternal loci, 11 paternal loci and 10 off-spring).

Mitochondrial DNA analysis

Genomic DNA extracted from tissue was used as a tem-plate for PCR amplification of the control region of themitochondrial DNA using three primer pairs, A and B, C(the complement of B) and D, and A and D (Fig. 2). Eachpair had one primer complimentary to sections of the con-served tRNA genes flanking the ends of the D-loop region,and one primer complimentary to a region around the ori-gin of replication, and have been previously used toamplify the control region in other species (e.g. eveningbats, Nycticeius humeralis, Wilkinson & Chapman 1991;cetaceans, Hoelzel, Hancock & Dover 1991). In selectedindividuals, the entire cytochrome-b gene was amplifiedfor sequencing using primers L14724 and H15915 (Irwin etal. 1991).

Double-stranded amplifications using PCR were car-ried out with a standard protocol using 10 ng of DNA in a25-µL reaction containing Taq buffer, 0.3 U of Taq poly-merase, and 78.5 pmol of each primer. After initially denaturing at 94 °C for 3 min, 35 cycles of 94 °C (30 s),45 °C (45 s), and 72 °C (45 s) were carried out. Negativecontrols were also included in each set of reactions. PCRproduct was purified using GENECLEAN® (BIO 101 Inc.,La Jolla, CA, USA).

Sequencing of double-stranded DNA products wasperformed using a Sequenase kit (United StatesBiochemical), and products were separated using a 6%polyacrylamide gel. Sequencing for the control region wasinitially carried out in both directions using primers A, B,C and D to obtain complementary partially overlapping

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

618 C . G . F A U L K E S E T A L .

strands. Subsequently, 11 additional primers (E–O inFig. 2) were identified and synthesized and used to obtainfurther overlapping sequence.

Control region sequences were obtained from a total of42 naked mole-rats (Table 1a), and two additionalBathyergid species for outgroup comparison (Cryptomysdamarensis and Heliophobius argenteocinereus). For each out-group, and for one representative H. glaber individualfrom Dembalawatchu, Lerata and Mtito Andei, the wholecontrol region was sequenced following PCR usingprimers A and D. Samples from the remaining individualswere amplified by PCR using primer pairs A and B, and Cand D, to give two nonoverlapping fragments, whichwhen sequenced produced 350–400 and 550 bp, respec-tively. It was only possible to obtain partial sequence datafor the formalin-fixed samples.

In addition, overlapping fragments of the cytochrome-bgene were sequenced in five representative naked mole-ratsamples, together with the two outgroup species (Table1b) using primers L14724 and H15915 (Irwin et al. 1991),and six internal primers (Fig. 2).

Analysis of mitochondrial DNA sequences

Sequences were aligned both manually using MacCladeversion 3 (Madison & Madison 1992) and with the aid of

the program C L U S T A L V (Higgins & Sharp 1989).Numbers of substitutions and transition/transversionratios were determined using MacClade. Relationshipsamong haplotypes were then analysed by a number ofphylogenetic algorithms, including maximum parsimonyusing bootstrap replications of the branch and boundsearch option in P A U P Version 3.1 (Swofford 1993), andbootstrap replications of the maximum parsimony byexhaustive search, maximum-likelihood, maximum-likeli-hood with molecular clock, U P G M A and neighbour-joiningmethods in P H Y L I P , version 3.57c (Felsenstein 1989).Kimura two-parameter genetic distances were calculatedwith the dnadist program in P H Y L I P .

The frequency of haplotypes and proportion ofsequence variation among colonies within regions, andamong regions was analysed using analysis of molecularvariance (A M O V A), a hierarchical approach analogous toanalysis of variance (Excoffier et al. 1992). A M O V A pro-duces estimates of variance components and F-statisticanalogues Φst, Φct and Φsc. A test for isolation by distancewhich examined the relationship between sequence diver-gence and geographical separation of haplotypes was performed using a Mantel test (N T S Y S Version 1.8; Rohlf1994)

Sequences have been deposited in NCBI with accessionnumbers U87521–U87538.

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 619

Fig. 2 Schematic of the control region and ajoining areas showing the relative positions of the primers used for PCR and sequencing inthis study. Primers listed above the diagram correspond to the light strand, while primers below the diagram correspond to the heavystrand. Sequences are as follows, together with the position of the 3′ end of the primer relative to the human mitochondrial DNA map for previously unpublished mole-rat specific primers (Anderson et al. 1981): (A) 5′-GAATTCCCCGGTCTTGTAAACC-3′ (L15926); (B) 5′-CCTGAAGTAGGAACCAGATG-3′; (C) 5′-CATCTGGTTCCTACTTCAGG-3′; (D) 5′-TCTCGAGATTTTCAGTGTCTTGCTTT-3′; (E) 5′-GATCAACATACCCCTCA-3′ (L16733); (F) 5′-CAGTTTGTTGTTTGTGTAG-3′; (L16574) (G) 5′-CCGTGAAACCATCAACCCGC-3′(L16322); (H) 5′-GTGCGATTAATAAGTGTTCG-3′ (L16174); (I) 5′-CAATTATATTTACACATAACG-3′ (L16374); (J) 5′-CCCCATAATA-AAAATCCCGGTAATG-3′ (L17049); (K) 5′-CGTCAACTCCCAAAGCTGAAATTC-3′ (L16006); (L) 5′-GCCCTTATCAAGGAGTGTAG-CAAAC-3′ (L16051); (M) 5′-TCAATATGACTATCCCCTTCCAATG-3′ (L16268); (N) 5′-GTTGTTTGTGTAGCACTGGTTTGTG-3′ (L16562);(O) 5′-GCGAGCTGAAATGTCATGGTTG-3′ (L16192); (H15915) 5′-AACTGCAGTCATCTCCGGTTTACAAGAC-3′; (MRL1) 5′-TAG-CACACATCTGCCGAG-3′ (L14960); (MRL3) 5′-TGAGGTGCTACCGTAATC-3′ (L15184); (MRL3HA) 5′-TGAGGAGCTACTGTGATT-3′(L15184); (MRL3HG) 5′-TGAGGTGCGACAGTCATC-3′ (L15184); (MRL4HG) 5′-CATTCCACCCCTACTACTC-3′ (L15420); (L14724) 5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′.

Results

Multilocus minisatellite DNA fingerprinting

Family analysis. In colony 2200 we analysed parents andtheir 13 offspring of one litter with minisatellite probe 33.6.A mean of 35.7 ± 0.6 bands were resolved for each animal.Banding patterns were very similar among individuals.Between parents, the coefficient of band sharing, x = 0.83.Among the offspring, x = 0.95 (n = 12 pairwise com-parisons). Segregation analysis of three paternal and ninematernal bands revealed a mean transmission frequency(between parents and offspring) of 49.4%, consistent withMendelian expectation. Kolmogorov–Smirnov goodnessof fit (D) values were 0.124 and 0.291 for maternal andpaternal cosegregation frequencies, respectively, and com-puter simulations of D indicated minimum values of ø of

0.50 for paternal bands (no linkage or allelism) and0.25–0.30 for maternal bands, at P < 0.05.

In colony N, the queen, three putative breeding malesand two litters of five offspring were analysed with probes33.6 and 33.15 (see Figs 3a and b). The mean number ofbands resolved were 32.5 ± 1.3 and 22.9 ± 0.9, respectively.Between the queen and breeding males, x = 0.81 ± 0.0006(n = 6 comparisons) and among the offspring, x = 0.81 ± 0.0003 (n = 45 pairwise comparisons). Segrega-tion analysis of three paternal and nine maternal bandsrevealed a mean transmission frequency (between parentsand offspring) of 48.1%, consistent with Mendelian expec-tation. Kolmogorov–Smirnov goodness of fit (D) valueswere 0.223 and 0.050 for maternal and paternal cosegrega-tion frequencies, respectively, and computer simulationsof D indicated minimum values of ø of 0.50 for paternalbands and 0.25–0.30 for maternal bands, at P < 0.05. This

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

620 C . G . F A U L K E S E T A L .

No. of No. basesLocation Colony Individuals sequenced Haplotype

(a)Somalia Som.1 2 200 Som1Ethiopia Eth. 1 4 950–1000 Eth1North Kenya Lerata 1 2 1000 Ler1North Kenya Lerata 2 5 1000 Ler2North Kenya Lerata 3 2 1000 Ler3North Kenya Lerata 4 4 1000 Ler1North Kenya Meru 1 2 570 Mer1SE Kenya Ithumba 1 2 1000 Ith1South Kenya Athi 1 1 1000 Ath1South Kenya Mtito Andei 6 4 1000 MAd1South Kenya Mtito Andei 1 7 350–1000 MAd1South Kenya Mtito Andei 2 3 900–1000 MAd1South Kenya Mtito Andei 4 1 930 MAd1South Kenya Mtito Andei 5 1 550 MAd1South Kenya Mtito Andei K 2 350–1000 MAd1Kenya Nairobi 1 1630 H.arg(Heliophobiusargenteocinereus)

Namibia D2 1 1630 C.dam(Cryptomysdamarensis)

(b)Ethiopia Eth. 1 1 1041 Eth1North Kenya Lerata 1 1 1127 Ler1North Kenya Lerata 2 1 1116 Ler2North Kenya Lerata 3 1 1071 Ler3South Kenya Mtito Andei K 1 1122 MAd1Kenya Nairobi 1 1137 Harg(Heliophobiusargenteocinereus)Namibia D2 1 1140 Cdam(Cryptomysdamarensis)

Table 1 Summary of samples, haplotype distribution and numberof bases sequenced for naked molerats and two outgroup speciesHeliophobius argentiocinereus andCryptomys damarensis for (a) themtDNA control region, and (b) themtDNA cytochrome-b gene

colony contained maximum heterozygosity and of thethree putative breeding males, two (96 and 98; Fig. 3a andb) contained unique bands which could be used diagnos-tically for paternity analysis. While male 99 could not beexcluded as father, it was possible to assign paternity ofindividuals in both of the litters to both males 96 and 98(Fig. 3a and b).

Local population analysis. Data from probes 33.6 and 33.15,which detect different sets of minisatellite loci, were com-bined to produce coefficients of band sharing (x) withinand between four wild colonies from the Mtito Andeiregion of southern Kenya (Table 2a). Intracolony co-efficients of band sharing, were higher in these naturalpopulations compared with the hybrid captive coloniespreviously described, with values ranging from 0.93 to 0.99.Values for x between closely adjacent colonies were alsohigh, ranging from 0.989 to 0.771, and although the samplesize was small, there was an apparent but nonsignificanttrend in decreasing x as the separation of coloniesincreased (from 1.0 to 5.0 km; r = 0.62; NS). In contrast to the Mtito Andei colonies, a high level of variability was found between colonies Lerata 2 and 4, with an

intercolony x = 0.29. This was a unique finding consider-ing the close position of these two colonies (separation was≈ 500 m).

Analysis of geographically distant populations

We compared banding patterns for probe 33.15 for indi-viduals from one colony from Ethiopia, two colonies fromLerata, northern Kenya, and two colonies from MtitoAndei, southern Kenya, and calculated values of x (Table2b). In the colony from Ethiopia, the intracolony coeffi-cient of band sharing of 0.99 was comparable to those ofcolonies at Mtito Andei. Intracolony values of x for Leratawere not calculated because too few animals could beanalysed. Individuals from Ethiopia and Mtito Andei, andLerata and Mtito Andei shared no common bands in thisstudy, while values for x between Ethiopia and the twoLerata colonies were very low (0.13 and 0.07; Table 2b).

Mitochondrial DNA D-loop sequences. For the 42 nakedmole-rats that were analysed at the control region, ninedistinct haplotypes were identified, one each from theEthiopian, Somalian and north Kenyan Meru colonies,

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 621

Fig. 3 DNA fingerprints for captive colony N from (a) minisatellite probe 33.6 and (b) probe 33.15, showing three putative breeding males(96, 98, 99), the breeding queen (97) and two litters of five offspring. One band unique to male 96 and three unique to male 98 were detect-ed (open and filled arrow heads, respectively). In both litters offspring could be unambiguously assigned to both of these breeding males.

three from the four colonies at Lerata, one each from thecolonies at Athi and Ithumba and one from the six coloniesat Mtito Andei (1, 2, 4, 5, 6 and K; Table 1; Fig. 1). For thefive individuals in which the entire control region wassequenced, insertion/deletion mutations were detected,particularly between the naked mole-rats and the twoother mole-rat species. With reference to the humansequence (Anderson et al. 1981) to determine the positionsof the more conserved flanking regions, and thereby determine the start and finish of the control region formole-rats, the sizes for the mole-rat control region were asfollows: naked mole-rat haplotypes: Ethiopia, 952 bp;Lerata 2 and Lerata 3, 962 bp; Lerata 1, 941 bp; Ithumbaand Athi, 945 bp; Mtito Andei 1, 963 bp; Heliophobius argen-teocinereus 1395 bp; Cryptomys damarensis 1383 bp. Amongthe naked mole-rats, the ratio of unambiguous transitionsubstitutions to transversions was 9.6. Aligned sequencesobtained for the nine naked mole-rat haplotypes showingthe 68 informative sites are shown in Table 3. A distancematrix quantifying the sequence differences betweenseven of the nine naked mole-rats (Somalian and Merusequences were incomplete and therefore omitted fromthe calculation) and the two outgroup haplotypes is

shown in Table 4. Among naked mole-rats, mean dis-tances ranged from 0.4% to a maximum of 4.6% betweenthe Ethiopian and the Kenyan haplotypes. The two otherspecies (H. argenteocinereus and C. damarensis) were toodivergent (> 50%) from naked mole-rats at this locus for ameaningful outgroup comparison, so they were notincluded in the phylogenetic analysis.

Phylogenetic analysis of sequences using maximumparsimony, maximum likelihood, maximum likelihoodwith molecular clock, neighbour joining and U P G M A allproduced trees having similar topology, with slight differ-ences only in the grouping of the Meru, Ethiopian andSomalian haplotypes relative to each other. A representa-tive cladogram from the distance based U P G M A method isshown in Fig. 4(a). The haplotypes Ler2 and Ler3 groupedtogether in all of the tree building analyses. Likewise, asecond major group containing haplotypes MAd1, Ith1,and Ler1 and Athi1 was consistently resolved. The nodessupporting both these groupings were supported by 100%bootstrap values (Fig. 4a). The three remaining haplotypesSom1, Eth1 and Mer1 either (i) formed a third distinctgroup with the Somalian and Ethiopian haplotypes togeth-er within it, or (ii) formed three independent lineages.

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

622 C . G . F A U L K E S E T A L .

Table 2 Mean ± SEM values for coefficient of band sharing (x) for n individuals from (a) four colonies from the Mtito Andei region ofKenya; (b) five colonies from from three different geographical locations Mtito Andei (south Kenya) Lerata (north Kenya) and Ethiopia(see Fig. 1); c = no of pairwise comparisons. Data are combined results for probes 33.6 and 33.15 (a) and probe 33.15 (b)

Colony K Colony 5 Colony 4 Colony 1(n = 5) (n = 1) (n = 3) (n = 5)

(a)Colony K x 0.99 ± 4.0 × 10–3 0.98 ± 2.0 × 10–5 0.90 ± 9.0 × 10–4 0.77 ± 4.0 × 10–5

c 10 5 15 25Colony 5 x – – 0.86 ± 5.0 × 10–5 0.81 ± 1.0 × 10–4

c 3 5Colony 4 x – – 0.93 ± 1.0 × 10–2 0.83 ± 5.0 × 10–3

c 3 15Colony 1 x – – – 0.97 ± 3.0 × 10–3

c 10

(b) Ethiopia Lerata Lerata Mtito Andei Mtito AndeiColony 1 Colony 2 Colony 4 Colony K Colony 1(n = 4) (n = 2) (n = 1) (n = 5) (n = 4)

Ethiopia x 0.91 ± 4.0 × 10–3 0.13 ± 4.0 × 10–3 0.07 ± 2.0 × 10–4 0.0 0.0Colony 1 c 6 8 4 16 16

Lerata x – – 0.29 0.0 0.0Colony 2 c 2 8 8Lerata x – – – 0.0 0.0Colony 4 c 4 4Mtito Andei x – – – 1.0 0.77 ± 1.0 × 10–5

Colony K c 6 16Mtito Andei x – – – – 0.96 ± 4.0 × 10–3

Colony 1 c 6

For cytochrome-b, a total of 1140 base pairs of sequence(corresponding to 379 amino acids) comprising the entiregene were obtained. Each of the seven samples (five nakedmole-rat and the two outgroups) that were sequenced pro-duced a distinct haplotype, and a distance matrix quanti-fying the differences between them is shown in Table 4.Amongst naked mole-rat haplotypes, similar patterns ofdivergence to those of the control region were also foundat the cytochrome-b gene, with mean sequence differencesamong naked mole-rats ranging from 0.5% to a maximumof 5.8% between the Ethiopian and the Kenyan haplo-types. At this locus, mean differences between nakedmole-rats and C. damarensis and H. argenteocinereus wereless than for the control region (30–35%, respectively),making sequence alignment and inclusion as outgroups inthe analysis possible. For phylogenetic analysis,cytochrome-b sequences were combined with the respec-tive control region sequences for five naked mole-rats andthe two outgroup species. In the case of the latter, becausethe control region sequences contained many inser-tions/deletions making complete alignment impossible,only 515 bp of the central conserved region were included.Maximum parsimony, maximum likelihood, maximumlikelihood with molecular clock, neighbour joining andU P G M A analyses all produced trees having identical topology. One tree was obtained from maximum parsimony

analysis and is shown in Fig. 4(b). The outgroup-rootedcombined cytochrome-b/control region data supports thecontrol region results shown in Fig. 4(a), with theEthiopian haplotype in a separate lineage to the othernaked mole-rat haplotypes, which group together as twosubclades containing Ler2 and Ler3, and Ler1 and MAd1,respectively. The tree in Fig. 4(b) also supports the position-ing of the outgroup H. argenteocinereus as phylogeneticallycloser to the naked mole-rat than C. damarensis, theDamaraland mole-rat. This observation is consistent withthe molecular phylogeny for the family Bathyergidaebased on sequence differences in the mitochondrial 12SrRNA gene (Allard & Honeycutt 1992).

We used A M O V A to contrast the molecular variance forcontrol region haplotypes (using the values in Table 3),both within and among colonies and among geographicallocations (Ethiopia, Lerata, Athi, Ithumba and MtitoAndei). As mentioned above, there was no variation inhaplotypes within any of the colonies studied, 68.3% of thevariance was explicable between geographical locations,and 31.7% between colonies within locations (e.g. atLerata).The Φ statistics generated by the analysis were asfollows: Φst = 1.00 (correlation of random haplotypes with-in a colony relative to all the haplotypes in the sample set);Φsc = 1.00 (correlation of random haplotypes within acolony relative to that within a regional grouping of

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 623

Table 3 Variable sites (68) for nine naked mole-rat control region haplotypes. Sequences are numbered from the 5′ end beginning at posi-tion 1 with reference to the human sequence (Anderson et al. 1981). Incomplete sequences were obtained from the Meru (Mer1) andSomalia (Som1) haplotypes, a ‘?’ denoting missing data. Only nucleotides that differ from the Ethiopian haplotype (Eth1) are shown, withdots (.) indicating that the base is the same as for Eth1. A dash (-) indicates an insertion/deletion mutation and has been added to main-tain alignment of the sequences.

colonies) and Φct = 0.68 (correlation of random haplotypeswithin a regional grouping of colonies relative to all thehaplotypes in the sample set). The values of 1.00 for bothΦst and Φsc reflects the fact that only one haplotype wasfound in each colony. The value of Φct = 0.68 was highlysignificantly different (P < 0.001) from a null distributionof Φct generated by 1000 permutations of the data set. Therelatively high value of Φct indicates that most of the vari-ation between haplotypes at the regional level was due todifferences between geographical regions rather thanwithin regions. The Mantel test results supplement thisobservation with a significant correlation betweensequence divergence and geographical separation of hap-lotypes (P = 0.004).

Discussion

Microgeographical structure: within colonies

In this study, we have attempted to investigate and quanti-fy the patterns of genetic structuring and divergence of

naked mole-rat populations at both the micro- and macro-geographical levels. Previous studies have shown thatwithin colonies of naked mole-rats in the wild, animals arehighly inbred, and loci that are normally highly polymor-phic lack variation (Faulkes et al. 1990b; Reeve et al. 1990;Honeycutt et al. 1991b). This observation makes certaintypes of genetic analysis (e.g. determination of parentage)difficult or impossible. The results obtained here frompedigree analysis of captive hybrid colonies formed frombreeders taken from geographically distinct areas ofKenya, have been shown to contain increased genetic vari-ation compared with pure-bred wild colonies, indicatingthe contribution of different alleles from northern andsouthern Kenyan populations. Segregation analysis of par-ents and offspring in these colonies gave results consistentwith Mendelian inheritance, assuming independent assort-ment and little or no linkage, thus validating this andprevious studies which have used multilocus fingerprint-ing to measure genetic variability and calculate relatednesswithin wild colonies of naked mole-rats (e.g. Faulkes et al.1990b; Reeve et al. 1990). Paternity analysis has shown for

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

624 C . G . F A U L K E S E T A L .

Eth1 Ler2 Ler3 Ler1 Ith1 Athi1 MAd1 H. arg C. dam

Eth1 – 4.9 4.8 4.8 NS NS 5.8 35.4 32.2Ler2 4.6 – 0.5 1.9 NS NS 3.0 33.9 30.6Ler3 4.6 0.8 – 2.1 NS NS 3.1 34.0 31.1Ler1 4.6 3.4 2.9 – NS NS 2.0 33.8 30.6Ith1 4.4 3.1 2.7 0.9 – NS NS NS NSAthi1 4.3 3.4 2.9 0.4 0.4 – NS NS NSMAd1 4.5 3.3 3.1 2.7 2.0 2.3 – 34.3 31.1H. arg 57.4 60.0 60.2 58.8 58.8 58.8 60.5 – 24.1C. dam 72.5 74.8 74.6 73.0 73.2 73.7 76.2 39.1 –

Table 4 Estimates of pairwisegenetic distances calculated fromKimura’s 2-parameter model inP H Y L I P (i) above diagonal:between five naked mole-ratcytochrome-b gene haplotypes andtwo outgroups (Heliophobius argen-teocinereus, H. arg, and Cryptomysdamarensis, C. dam; NS, sample notsequenced); (ii) below diagonal:between seven of nine naked mole-rat haplotypes for which thewhole control region wassequenced, and two outgroups

Fig. 4 Cladograms showing phylogeneticrelationships of nine naked mole-rat haplotypes and two outgroup species(Heliophobius argenteocinereus, Harg, andCryptomys damarensis, Cdam): (a) rootedtree generated from U P G M A analysis ofcontrol region sequences; (b) single treegenerated from maximum parsimonyanalysis of combined control region andcytochrome-b sequences, rooted using theoutgroups, using the branch and boundsearch option in P A U P (tree length 767,consistency index 0.948, homoplasy index0.052). Numbers above each branch referto the percentage bootstrap values follow-ing 100 replications, while values below initalics are branch lengths.

the first time, at least in this hybrid captive ‘colony N’, thatlitters may be sired by more than one breeding male. Theadaptive significance of multimale parentage in nakedmole-rats and the criteria that determine who becomes abreeding male remain unclear, although these males tendto have a higher dominance rank within the colony hierar-chy (Faulkes & Clarke, unpublished; Schieffelin & Sherman1995). Given the high genetic similarity of colony members,it is unclear what benefit the queen gains by mating withmore than one male. Two possible explanations are (i) thatthere is a small amount of residual genetic variation with-in colonies and the queen may maintain some variability inher offspring and reduce the effects of inbreeding by mat-ing with more than one male (some DNA fingerprintsshow small differences between individuals within acolony – see results), possibly by selecting mates on thebasis of small differences at the major histocompatibilitycomplex (MHC) (Potts et al. 1991); (ii) as naked mole-rats areso highly inbred, it may be that mating with more than onemale increases conception rates. As in some other inbredspecies, the sperm of naked mole-rats are highly polymor-phic in shape and size (C. G. Faulkes, unpublished data),and inbreeding, a high incidence of structurally abnormalsperm, and reduced fertility have been linked in a numberof species (e.g. lions. Wildt et al. 1987). Choosing to matewith males that have heritable dominant traits would onlyhave a fitness benefit for the queen if mixing of coloniesand/or dispersal occurs in the wild and her offspring maytherefore have an increased chance of reproduction andgreater reproductive success. Previously, dispersion andoutbreeding were thought to be extremely rare in the wild(Sherman et al. 1992), although the recent observation ofsome male dispersal in captive colonies of naked mole-ratsshowed that such events can occur (O’Riain et al. 1996).

Microgeographical structure between colonies

The genetic composition of neighbouring colonies in the wildmay have important consequences for dispersal events,and in this study we have identified variable patterns ofgenetic structuring of colonies within geographical areas.At Mtito Andei intercolony genetic variability was low,with all six colonies sharing the same control region hap-lotype, and having relatively high coefficients of bandsharing in minisatellite DNA fingerprints. Low geneticvariability among colonies at Mtito Andei has been report-ed previously, following RFLP analysis of mitochondrialDNA (Honeycutt et al. 1991b). The occurrence of one hap-lotype in the colonies investigated in this study impliesthat they are all derived from a common maternal ances-tor distinct from that of the relatively close populations atAthi and Ithumba. The genetic similarity of the MtitoAndei colonies makes it difficult to determine, using thesemolecular techniques, whether or not any dispersal events

have occurred. In the case of the Mtito Andei colonies, dis-persal might be expected to be beneficial for both the disperser, who might gain an increase in direct fitness, andthe recipient colony, which would gain a small increase ingenetic variation. At the same time, the cost in terms ofloss in inclusive fitness to nonbreeders within the recipientcolony if the disperser breeds should be limited becausethese nonbreeders would also share a high proportion ofalleles in common with the disperser.

In contrast, at Lerata two colonies (Lerata 1 and 4)shared the same haplotype (‘Ler1’) which grouped in thesame clade as the southern Kenyan colonies in the phylo-genetic analysis. These two colonies were quite divergentfrom the other two haplotypes (found in colonies Lerata 2and 3, respectively), which only had a low level of diver-gence between them. These differences were also reflectedin the coefficients of band sharing from the DNA finger-prints, despite the fact that these colonies were in closeproximity (Table 2b; Fig. 1). It is possible that thesecolonies are at the boundary of two distinct populations,one of which is derived from southern Kenya. Theseresults are supported by allozyme studies of Lerata ani-mals, which revealed that one out of 34 loci showed heterozygosity among colonies, in contrast to Mtito Andeiwhere individuals were homozygous for all 34 loci(Honeycutt et al. 1991b). Furthermore, mtDNA RFLPanalysis of individuals from Samburu, a region just south-east of Lerata, also showed increased intercolony variationcompared with Mtito Andei (Honeycutt et al. 1991b). Thus,the costs and benefits of dispersal at Lerata are potentiallydifferent to the Mtito Andei location. Because of the high-er genetic heterogeneity, a greater fitness loss would beincurred among the nonbreeding helpers of a colonyaccepting a male immigrant, because the subsequent off-spring of the queen and the new male would be less related to the nonbreeders. From the data collected in thisstudy it is difficult to say which is the more typical pattern.More sampling of multiple colonies at different locationswould be required to resolve this issue. However, the sis-ter group relationships between the MAd1, Ith1, Athi1 andLer1 haplotypes is suggestive of an isolation by distancemodel of divergence where ‘budding’ of colonies and lim-ited dispersal and gene flow produced haplotypes thatincrease in sequence difference with increasing geograph-ical distance. The extent to which naked mole-rats are ableto discriminate one another on a genetic basis is also atpresent untested, and until these questions are addressedthe exact costs and benefits of dispersal in terms of individ-ual and inclusive fitness gains and losses will not be clear.

Macrogeographical structure

Mean genetic distances calculated from pairwise compar-isons of control region sequences produced very similar

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 625

values to the cytochrome-b gene sequences (Table 4). Thiscontrasts with other species where the control region hasbeen shown to be more variable than coding regions (e.g.the human: Aquadro & Greenberg 1982). The divergencein control region sequences between the naked mole-ratand the two outgroup species, C. damarensis and H. argen-teocinereus, and the larger size of the sequence in the outgroups, is consistent with other molecular evidence suggesting that naked mole-rats are highly divergent fromother species in its family (C.G. Faulkes et al. unpubl. data;Allard & Honeycutt 1992). Phenotypically, naked mole-rats have a mating system promoting extreme inbreeding,in contrast with the eusocial C. damarensis, which has anincest avoidance mechanism (Jarvis & Bennett 1994), andmorphological traits such as small body size, elongatedtail and absence of a pelage, which are unique traits com-pared with other bathyergids.

Results from both multilocus minisatellite finger-printing and sequence analysis of the two mitochondrialDNA loci have shown that at a macrogeographical level,colonies of naked mole-rats show considerable diver-gence, with fixation of different genotypes occurring at thedifferent geographical locations. DNA fingerprints andmtDNA haplotypes of samples collected from populationsin Ethiopia, Somalia and Meru in northern Kenya weredistinct from those from Lerata in northern Kenya, andMtito Andei, Athi and Ithumba in southern Kenya. Thesegregation of mtDNA haplotypes within distinct areasprobably reflects the high population viscosity (i.e. limiteddispersal) and limited gene flow imposed by the subter-ranean niche of the mole-rat, and the patchiness of suitablehabitats that can occur within an area (J. U. M. Jarvis, per-sonal communication).

From the sequence data, the mean genetic differenceswere maximum between the Ethiopian and southernKenyan Mtito Andei haplotypes at 5.8% for thecytochrome-b gene. In vertebrates, mtDNA is cited as hav-ing an approximate divergence rate of 2% per millionyears (Moritz et al. 1987), while Krajewski & King (1996)calculated a divergence rate of 0.7–1.7% per million yearsfor the cytochrome-b gene of cranes. Assuming an approx-imate divergence rate of 2% per million years for the mole-rat cytochrome-b gene, then this would put the timesince the Ethiopian and Mtito Andei individuals shared acommon ancestor at ≈ 2.9 million years ago. The fossilrecord of naked mole-rats in east Africa is known toextend back approximately 3 million years (VanCouvering 1980), suggesting an ancient divergencebetween Ethiopian and Kenyan populations. The value of5.8% divergence between Ethiopian and southern Kenyanhaplotypes is slightly greater than the intraspecific rangefor cytochrome-b genetic distances known for otherAfrican mole-rats (Cryptomys damarensis Namibian vs.Botswana populations, approx. 700 km separation, 1.5%;

Cryptomys hottentotus Cape Town vs. Klawer populationsin South Africa, approx. 230 km separation, 4.2%; C.G.Faulkes et al. unpubl. data), and approaching that of someinterspecific distances (Bathyergus suillus vs. Bathyergusjanetta, 5.9%; Cryptomys damarensis vs. Cryptomys hottento-tus amatus, 6.6%; C.G. Faulkes et al. unpubl. data). At themacrogeographical level, there was a clear trend for isola-tion by distance, with absolute geographical distancebeing significantly correlated with sequence divergence.As a consequence of this, and other factors like long-termbarriers to gene flow and limited female dispersal, widelydispersed naked mole-rat populations are highly diver-gent, although the available evidence suggests thatH. glaber is still a monotypic species. Certainly, animalsfrom colonies Lerata 2 and 3 will readily breed with MtitoAndei individuals in captivity, despite genetic distances of3% between them at the cytochrome-b gene, although it isnot known whether this would also hold true forEthiopian and Somalian individuals. The low speciesdiversity of naked mole-rats is surprising given the highdegree of genetic differentiation of conspecific populationswhen compared with other bathyergids, and the ancientancestry of the naked mole-rat within the family (Allard &Honeycutt 1992). In contrast, the sister genus Cryptomys,which inhabits a wider range of habitats, is relativelyspecies rich, with at least 13 taxa with a variety of socialstructures (C.G. Faulkes et al. unpubl. data). While wehave sampled individuals over a large proportion of therange of the naked mole-rat, populations in the far north-ern and eastern extremes in Ethiopia and Somalia remainto be investigated. Differences in the lengths of chromoso-mal short arms have been previously noted betweenKenyan and Somalian individuals (Honeycutt et al. 1991a).Phylogenetic analysis on a macrogeographical level ofnaked mole-rat mtDNA haplotypes has not revealedwhich are ancestral forms, thereby preventing us fromtesting the hypothesis that mole-rats spread into their cur-rent east African range from south-western Africa throughan ‘arid corridor’ (Honeycutt et al. 1991a).

In summary, our results suggest that fission of highlyinbred colonies coupled with rare male dispersal events,form local populations that are genetically similar, withdivergence increasing with distance (e.g. Mtito Andei,Athi and Ithumba populations), or maintenance of highlydivergent matrilines within a small area (e.g. Lerata).Ultimately, the limits to gene flow imposed by the sub-terranean niche has led to populations with sequencedivergence approaching interspecific levels.

Acknowledgements

We gratefully thank the following for supplying some of the tis-sue samples used in this study: Professor J. U. M. Jarvis,University of Cape Town and Dr N. C. Bennett, University

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

626 C . G . F A U L K E S E T A L .

of Pretoria (H. glaber from Lerata, and C. damarensis and H.argenteocinereus.); Mike Jacobs and Cathy Schloeder (Ethiopiansamples); Karl Kranz, Zoological Society of Philadelphia (Merusamples), Gary Bronner, Transvaal Museum Pretoria (Somaliansamples), and Professor J. U. M. Jarvis, for also supplying live animals (colony 2200 and the ancestors of colony N). We areindebted to Mark Beaumont for advice on data analysis, DebbieSmith for assistance in the laboratory, Terry Dennett for photo-graphy and Mandy Gordon and Mike Llovet for care of our captive colonies of naked mole-rats, and Shaun Faulkes for assis-tance with the field work. We would like to express our thanks forpermission to carry out the field work to the Office of thePresident and the Ministry of Tourism and Wildlife, Republic ofKenya. This work was funded by the Institute of Zoology andresearch grants to C. G. Faulkes from the Bonhote Bequest of theLinnean Society and the Royal Society, and travel grants from theRoyal Society, the Wellcome Trust, the Society for Endocrinology,JRF Ltd and the Institute of Zoology (to D. H. Abbott) and theSERC and JRF Ltd (to C. G. Faulkes). This is publication number36–056 of the Wisconsin Regional Primate Research Center.

References

Allard MW, Honeycutt RL (1992) Nuceotide sequence variation inthe mitochondrial 12S rRNA gene and the phylogeny ofAfrican mole-rats (Rodentia: Bathyergidae). Molecular Biologyand Evolution, 9, 27–40.

Anderson S, Bankier AT, Barrell BG et al. (1981) Sequence andorganization of the human mitochondrial genome. Nature,London, 290, 457–465.

Aquadro CF, Greenberg BD (1982) Human mitochondrial DNAvariation and evolution: analysis of nucleotide sequences fromseven individuals. Genetics, 103, 287–312.

Brett RA (1991) The population structure of naked mole-ratcolonies. In: The Biology of the Naked Mole-Rat (eds ShermanPW, Jarvis JUM, Alexander RD), pp. 97–136. PrincetonUniversity Press, New York.

Bruford MW, Burke T (1994) Minisatellite DNA markers in thechicken genome. I. Distribution and abundance of mini-satellites in chicken multilocus DNA fingerprints. AnimalGenetics, 25, 381–389.

Bruford MW, Hanotte O, Brookfield JFY, Burke T (1992) Singleand multi-locus DNA fingerprinting. In: Molecular GeneticAnalysis of Populations: a Practical Approach (ed. Hoelzel AR),pp. 225–269. IRL Press, Oxford.

Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecularvariance inferred from metric distance among DNA haplo-types: application to human mitochondrial DNA restrictiondata. Genetics, 131, 479–491.

Faulkes CG, Abbott DH, Jarvis JUM (1990a) Social suppression ofovarian cyclicity in captive and wild colonies of naked mole-rats, Heterocephalus glaber. Journal of Reproduction and Fertility,88, 559–568.

Faulkes CG, Abbott DH, Jarvis JUM (1991) Social suppression ofreproduction in male naked mole-rats, Heterocephalus glaber.Journal of Reproduction and Fertility, 91, 593–604.

Faulkes CG, Abbott DH, Mellor A (1990b) Investigation of geneticdiversity in wild colonies of naked mole-rats by DNA finger-printing. Journal of Zoology, London, 221, 87–97.

Felsenstein J (1989) PHYLIP – Phylogentic Inference Package,Version 3. 2. Cladistics, 5, 164–166.

Hamilton WD (1964) The genetical evolution of social behaviour.I, II. Journal of Theoretical Biology, 7, 1–52.

Higgins DG, Sharp PM (1989) Fast and sensitive multiplesequence alignments on a microcomputer. Cabios, 5, 151–153.

Hoelzel AR, Hancock JM, Dover GA (1991) Evolution of thecetacean mitochondrial D-loop region. Molecular Biology andEvolution, 8, 475–493.

Honeycutt RL, Allard MW, Edwards SV, Schlitter DA (1991a)Systematics and evolution of the family Bathyergidae. In: TheBiology of the Naked Mole-Rat (eds Sherman PW, Jarvis JUM,Alexander RD), pp. 45–65. Princeton University Press, New York.

Honeycutt RL, Nelson K, Schlitter DA, Sherman PW (1991b)Genetic variation within and among populations of the nakedmole-rat: Evidence from nuclear and mitochondrial genomes.In: The Biology of the Naked Mole-Rat (eds Sherman PW, JarvisJUM, Alexander, RD), pp. 195–208. Princeton University Press,New York.

Irwin DM, Kocher TD, Wilson AC (1991) Evolution of thecytochrome-b gene of mammals. Journal of Molecular Evolution,32, 128–144.

Jarvis JUM (1981) Eu-sociality in a mammal – cooperative breed-ing in naked mole-rat Heterocephalus glaber colonies. Science,NY, 212, 571–573.

Jarvis JUM (1991) Reproduction of naked mole-rats. In: TheBiology of the Naked Mole-Rat (eds Sherman PW, Jarvis JUM,Alexander RD), pp. 384–425. Princeton University Press, NewYork.

Jarvis JUM, O’Riain MJ, Bennett NC, Sherman PW (1994)Mammalian eusociality: a family affair. TREE, 9, 47–51.

Jeffreys AJ, Wilson V, Thein SL (1985a) Hypervariable ‘mini-satellite’ regions in human DNA. Nature, London, 314, 67–73.

Jeffreys AJ, Wilson V, Thein SL (1985b) Individual-specific‘fingerprints’ of human DNA. Nature, London, 316, 76–79.

Krajewski C, King DG (1996) Molecular divergence and phylo-geny: Rates and patterns of Cytochrome b evolution in cranes.Molecular Biology and Evolution, 13, 21–30.

Lacey EA, Sherman PW (1991) Social organization of naked mole-rat colonies: Evidence for a division of labor. In: The Biology ofthe Naked Mole-Rat (eds Sherman PW, Jarvis JUM, AlexanderRD), pp. 275–336. Princeton University Press, New York.

Lovegrove BG, Wissel C (1988) Sociality in mole-rats: metabolicscaling and the role of risk sensitivity. Oecologia, 74, 600–606.

Madison WP, Madison DR (1992) MacClade: analysis of phylogenyand character evolution, version 3. Sinauer Associates, Inc., MA,USA.

Meester JAJ (1960) The Col. Jack Scott Somalia Expedition. BulletinTrans. Mus. No.4, 5–8.

Moritz C, Dowling TE, Brown WM (1987) Evolution of animalmitochondrial DNA: relevance for population biology andsystematics. Annual Revue of Ecology and Systematics, 18,269–292.

O’Riain MJ, Jarvis JUM, Faulkes CG (1996) A dispersive morph inthe naked mole-rat. Nature, 380, 619–621.

Potts WK, Manning CJ, Wakeland EK (1991) Mating patterns inseminatural populations of mice influenced by MHC geno-type. Nature, 352, 619–621.

Reeve HK, Westneat DF, Noon WA, Sherman PW, Aquadro CF(1990) DNA ‘fingerprinting’ reveals high levels of inbreedingin colonies of the eusocial naked mole-rat. Proceedings of theNational Academy of the USA, 87, 2496–2500.

Rohlf FJ (1994) Numerical taxonomy and multivariate analysissystem. Exeter Software, New York.

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

G E N E T I C S T R U C T U R I N G O F N A K E D M O L E - R A T C O L O N I E S 627

Schieffelin JS, Sherman PW (1995) Tugging contests reveal feed-ing hierarchies in naked mole-rat colonies. Animal Behaviour,49, 537–541.

Sherman PW, Jarvis JUM, Braude SH (1992) Naked mole-rats.Scientific American, 267(2), 42–48.

Swafford DL (1993) PAUP: Phylogenetic Analysis Using Parsimony(Version 3.1). (Computer Program). Illinois Natural HistorySurvey, Champaign.

Van Couvering JAH (1980) Community evolution in East Africaduring the late Cenozoic. In: Fossils in the Making: VertebrateTaphonomy and Paleocecology (eds Behrensmeyer AK, Hill AP),pp. 272–298. Chicago University Press, Chicago.

Wildt DE, Bush M, Goodrowe KL et al.(1987) Reproductive andgenetic consequences of founding isolated lion populations.Nature, 329, 328–331.

Wilkinson GS, Chapman AM (1991) Length and sequence

variation in evening bat D-loop mtDNA. Genetics, 128,607–617.

Wilson EO (1971) The Insect Societies. Belknap Press of HarvardUniversity Press, Cambridge MA.

This paper is part of a long-term study on the genetics of Africanmole-rats by C. Faulkes and M. Bruford, started in 1987 by D.Abbott and C. Faulkes. The aim of our project is to use moleculargenetic techniques, in conjunction with ecological data collectedby ourselves and our collaborators in South Africa (J. Jarvis andN. Bennett), to tackle questions related to the evolution of sociali-ty using the family Bathyergidae as a model system. R. Waynehelped in the development of the project, and some of the earlymethodological development and sequencing work was carriedout with the help of M. Roy, L. Lau and H. O’Brien.

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 615–628

628 C . G . F A U L K E S E T A L .