Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

Evlolecular Ecology (1994) 3,301-312

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis D. GOTTELLI,* C. SILLERO-ZUBIR1,t G. D. APPLEBAUM,S M. S. ROY,* D. J. GIRMAN,S J. GARCIA-MORENO, $ E. A. OSTRANDERS and R. K. WAYNE"$ *Institute of Zoology, Zoologicnl Society of London, Regent's Park, London NWI 4RY, UK, tWildlife Conservation Research Unit, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK, $Department of BioZogy, University of California, Los Angeles, CA 90024, U S A and §Fred Hutchinson Cancer Research Centre, Mailstop M318, 1124 Coliirnbin St., Senttlr, W A 98104, U S A

Abstract

The world's most endangered canid is the Ethiopian wolf Canis simensis, which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibiIity through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.

Keywords: canids, endangered species, hybridization, microsatellite, mitochondrial DNA, systematics

Received 9 September 1993; revision received 12 [ a n u a y 1994; accepted 17 Jnniiary 2994

Introduction

The Ethiopian wolf, more commonly known as the Simien jackal, Canis simensis, is a coyote-size, (11 to 19 kg) highly endangered canid found only in the highlands of Ethiopia. It is also referred to less frequently as the Simien fox or Abyssinian wolf. Fewer than 500 individuals occur in six isolated populations on both sides of the Ethiopian

Correspondence: R. K. Wayne, Institute of Zoology, Zoological Society of London, Regent's Park, London NW14RY, UK. Fax 071 586 2870.

Rift Valley and are restricted to Afro-alpine habitat above 3000 metres where they prey exclusively on rodents (Gottelli & Sillero-Zubiri 1992; Yalden & Largen 1992). The largest population is located in the Bale Mountains (Fig. 1). Ethiopian wolves are very distinctive, having a reddish coat, with white underparts, throat, chest and tail markings. They are territorial, social animals, living in multi-male packs observed to be as large as 13 adults. Only one female per pack breeds annually, all males re- main in the natal pack and often daughters replace their mothers as breeders, suggesting the possibility of inbreeding (Gottelli & Sillero-Zubiri 1990). However, two

302 D. G O T T E L L I rt a[.

Mounluns

Condcr

T

Addis Ababa

Arri 0

Fig. 1 Map of sampling localities indicating extant populations of Ethiopian wolves and area sampled in the present study.

behaviours that we have observed may counterbalance limited inbreeding: first, some 2-year-old females dis- perse; secondly, the dominant female may also mate with males from neighbouring packs (Sillero-Zubiri & Gottelli unpublished data).

Although regarded as an extremely endangered species, the Ethiopian wolf has been little studied, and is not currently bred in captivity (Ginsberg & Macdonald 1990). Numbers in the wild are decreasing due to habitat de- struction associated with agriculture, overgrazing and in- creasing human populations (Yalden 1983; Sillero-Zubiri & Gottelli 1991). Other significant threats are abundant domestic dogs that may hybridize with Ethiopian wolves and carry diseases such as rabies (Mebatsion ef al. 1992) and compete with them for food. For example, between 1990 and 1992, the Bale Mountain population was halved by an outbreak of rabies. In response to a questionnaire given to 60 Bale Mountain shepherds, three individuals reported male domestic dogs mating with Ethiopian wolves (Gottelli & Sillero-Zubiri 1990). Field observations of 156 animals in the Bale Mountain National Park found 8% had abnormal coat colour (Sillero-Zubiri & Gottelli 1991; Gottelli & Sillero-Zubiri 1992). Ethiopian wolves are

also the victims of automatic weapons that proliferated during the recent civil war. The Ethiopian Highlands are among the most densely populated agricultural areas in Africa; the Afro-alpine grasslands are increasingly used for grazing, and Ethiopian wolves cannot easily avoid contact with humans and their domestic animals.

In 1988, the New York Zoological Society initiated an ecological study of Ethiopian wolves in the Bale Moun- tain National Park. The study focused on populations from the Sanetti Plateau and Web Valley, two areas sepa- rated by 20 km of inhospitable rocky peaks, crossed by narrow corridors of suitable habitat (Fig. 1). Feral domestic dogs were abundant in Web Valley but nearly absent from Sanetti. Blood samples were obtained from Ethio- pian wolves from both localities and from domestic dogs, and molecular genetic techniques were used to address four questions of conservation importance: 1 how phylogenetically distinct are Ethiopian wolves from other wolf-like canids; 2 is there genetic evidence for hybridization between domestic dogs and Ethiopian wolves; 3 i s the genetic variability of Ethiopian wolves low rela- tive to other wolf-like canids; and 4 are the two closely situated Ethiopian wolf populations genetically isolated?

We analysed variation in nuclear and mitochondrial DNA to address these four questions. First, we used DNA sequence and restriction fragment analysis of the mitochondrial genome to determine phylogenetic affinities, genetic variability and hybridization in Ethiopian wolves. The mitochondrial genome in mammals is usually mater- nally inherited without recombination, and has a higher rate of DNA sequence evolution than most nuclear genes (Brown 1985). Consequently, analysis of the mitochondrial genome can provide clues to the phylogenetic relationships of closely related taxa and evidence for hybridization between female domestic dogs and male Ethiopian wolves. Moreover, mtDNA variation will be lost rapidly in small populations because the effective size of the mitochondrial genome is approximately one quarter that of nuclear genes. Therefore, its analysis may be a sensitive indicator of genetic loss in small populations (Avise et al. 1988).

Secondly, we assessed polymorphism in the class of hypervariable nuclear loci known as microsatellites. These loci consist of repeats of a simple nucleotide sequence that evolve through the gain or loss of repeat units rather than sequence substitutions. Microsatellite loci are highly polymorphic with frequently more than a dozen alleles at a single locus, have high mutation rates exceed- ing lo4 per generation and are widely dispersed in eukaryotic genomes (Tautz et nl. 1986, Tautz 1989; Weber & May 1989; Litt & Luty 1989; Heame et 01. 1991; Moore ef al. 1991; Dietrich ef nl. 1992; Ostrander ef n f . 1993). Due

MOLECULAR GENETICS OF THE MOST ENDANGERED CANID 303

to the high polymorphism and evohtionary rate of microsatellite loci, they are potentially very informative with regard to analysis of genetic differentiation and hybridization.

Methods

Sample localities

We analysed mitochondrial and microsatellite variation from the Sanetti Plateau and Web Valley Ethiopian wolf populations, totalling 54 individuals. Blood samples were obtained during two summer field seasons in 1989 and 1991 (Table 1). During the first field season, three social groups totalling 14 individuals were sampled from the Sanetti population (B-3, C-2, D-9), and three social groups, totalling nine individuals, were sampled from the Web Valley population (H-3, S-3, W-3) (Table 1). DNA isolated from blood samples of these individuals was analysed for mitochondrial restriction fragment length variation. Part of the mitochondrial DNA control region I (394 bp) was sequenced from two individuals belonging to one social group from Sanetti (B3 and 87) and six individuals from three social groups in Web Val- ley (H3, H5, S5, W2, and W7). This region may be hypervariable in vertebrates and provide more resolution among closely related populations than studies of more conserved genes (Vigilant et al. 1991; Wenink ei 01. 1993).

We also studied, by restriction site analysis, an un- associated individual (Di) thought to be a dog-Ethiopian-

Table 1 Sampling localities, year, pack affiliations and individual Ethiopian wolves sampled. Brackets enclose littermates. Stars indicate phenotypically abnormal individuals (see text)

Pack/ Locality Year breeds Individuals

Sanetti 1989 B C D

1991 B C D N

WebValley 1989 H S W

Domestic dogs 1991 F

P S T w

Dog breeds 32

1,3,7 3,7 1,4,5,6,7,8,9,10,12 1,2,3,7 [6,13] [10,12,17,19] 4,9 4,5 2,14,17,19 3 7 3 1,2,5* 2,5.7* Di* 1,4.5,6,7,8,9 5,7,8,9,11,3 2 11,13 2,3',5' [4,8,10,11] 2 [5',6,7*] [8*,9*,10,11*] 40 individuals

wolf hybrid, and seven domestic dogs from Web Valley (Table 1). Additionally, Di and two of these domestic dogs (4 and 7, Table 1) were analysed for variation in control region sequence. Except for the sample from domestic dog 4 and a minute quantity of DNA from Di, all DNA samples from the first field season were entirely depleted by the mtDNA analyses and consequently couId not be analysed for microsatellite variation.

A second group of 41 Ethiopian wolves was collected in Summer, 1991 and included samples from nine social groups, four in Sanetti (B-10, C-2, D-2, N-4) and five in Web Valley (F-6, P-2,57, T-1, W-7). In both years, five of the same Ethiopian wolves were caught in Sanetti and four in Web Valley. Seven Ethiopian wolves in Web Val- ley packs were identified as phenotypically aberrant and thought to be dog-Ethiopian-wolf hybrids (Table 1). Ab- normal individuals were obvious and identified by their unusual coats, kinked tails, loss or changes in white marking. One Web Valley dog from 1989 (4, Table 1) and all Ethiopian wolves from 1991 were analysed for variation in nine microsatellite loci.

Domestic dogs and other canids

We examined microsatellite variation in forty blood samples from domestic dogs representing .32 dog breeds to identify dog specific alleles. These samples had been analysed for mitochondrial DNA restriction fragment variation as listed in Wayne et al. (1992).

DNA isolated from blood or tissue samples of other canids were available from past population surveys of wolf-like canids (Wayne et al. 1990 1991 1992; Lehman eta[. 1991; Wayne & Jenks 1991; Girman et al. 1993). We analysed 1540 individuals from five wolf-like canid species for microsatellite variation, including gray wolves Canis lupus, coyotes C. latrans, golden jackals C. atfretis, and black-backed jackals C. mesometas (Roy et al. in press). Two individuals from these species and the Afri- can wild dog Lycaon pictus, the side-striped jackal C. adustiis, and the gray fox Urocyon cinereoargenteus were analysed for the mtDNA sequence analysis.

Mitochondria1 D N A restriction site analysis

Genomic DNA was isolated by proteinase K digestion, extracted with phenol-chloroform-isoamyl alcohol, eth- anol precipitated and resuspended to yield a final concen- tration of about 1 mg/mL (Sambrook e ta l . 1989). Ap- proximately 5 rng of DNA from each sample was digested separately with an excess of 17 restriction endonucleases: AccI, BamHI, BclI, BglI, BgllI, ClnI, Drat EcoRI, EcoRV, HhaI, W i n d , HindlU, Hpal, NcoI, SstI, Stril and XmaI. The digested DNA was then electrophoresed through a 1% agarose gel, transferred to a nylon mem-

304 D. GOTTELLI et al.

brane by capillary blotting, and probed using cloned radiolabelled mtDNA from a domestic dog (Wayne et al . 1990). Autoradiography was used to examine patterns of migration of mtDNA fragments. The fragments were sized by comparison with molecular weight standards (bDNA digested with HindIII) and summed to assay for length mutations in the mtDNA genome. A mitochondria] DNA genotype is defined by the composite restriction fragment patterns for each individual for the 17 restriction enzymes (Lehman ef al. 1991).

D N A Sequencing

The dideoxynucleotide chain termination method was used to sequence 394 bp of the mitochondria1 control region I, following amplification by the polymerase chain reaction (PCR) (Sambrook et al. 1989). Primer sets for these regions were based on universal primers used in studies of vertebrates and include L15905 (5'-TAATACA- CCAGTC?"TGTAAACC-3') and H16517 (5'-CCTGAAG- TAGGAAC CAGA-33 (Anderson et al. 1981; Kocher et al. 1989). We also sequenced a total of 2001 bp from three protein coding genes, cytochrome b, and cytochrome oxidase I and I1 (COI, COII), from two Ethiopian wolves to assess their phylogenetic relationships to other wolf-like canids. Primer sets for these regions were based on universal PCR primers and include: cytochrome b - H15149

CTCA-3') (Kocher et al. 1989), L14724 (5'-CGAAGCTTG-

AGACCCTGACAACTA-3'), and H15915 (5'-AACTGC- AGTCATCrCCGGT'ITACAAGAC-3'), (Kocher et a!. 1989; Irwin etal. 1991; Girman etal. 1993); cytochrome oxidase I - L6569 (5'-CCTGCAGGAGGAGGAGATCC- 3') and H7227 (5'-AGTATAAGCGTCTGGGTAGTC-3'); and cytochrome oxidase I1 - L7552 (5'-AACCATTTCAT- AACTTGTCAA-3') and H8321 (5'-CTCTTAATCTlTA- ACTTAAAG-3') (Anderson et al. 1981). Each PCR reaction mixture contained approximately 100 ng of genomic DNA; a reaction buffer of 50 mM KC1, 2.5 mM MgCI,, 10 mM Tris-HC1 (pH 8.8), 1 mM dNTP mix, and 2-2.5 units of Ta9 DNA polymerase (Promega) in a volume of 50 mL. We used 25 pmoles of each primer and a Perkin-Elmer Cetus DNA thermocycler programmed for 35 amplification cycles with denaturation at 94 "C for 45 s, annealing at 50 "C for 30 s, and extension at 72 "C for 45 s. Double- stranded reaction products were fractionated by electro- phoresis using 3% Nusieve agarose (FMC corporation, Rockland, MD). The appropriate size band was excised, then purified by Geneclean (BIO 101, La Jolla, California), and the products were sequenced using a Sequenase kit (US Biochemical). Sequence data was deposited in GenBank (Accession no. L2941SL29416). Sequence divergence estimates were corrected for multiple hits (Jukes

(5'-AAACTGCAGCCCCTCAGAATGATATTTGTC-

ATATGAAAAACCATCG'ITG-3'), L15513 (5'-CTAGG-

& Cantor 1969) and a standard error calculated using the approach of Nei et al. (1985).

To analyse sequence data, we chose an unweighted maximum parsimony approach, with the gray fox as an outgroup. The PAUP version 3.1.1 for the Apple Macintosh computer was used to determine the most parsimonious trees with the branch and bound option (Swofford 1990). The statistical confidence of each node was judged by as- sessing the frequency of nodes supported in 1000 bootstrap resamplings of our data (Felsenstein 1985). The gl statistic was calculated to judge the significance of the phylogenetic signal provided by our sequence data (Hillis & Huelsenbeck 1992).

As an alternative phylogenetic approach, we used the maximum likelihood program (DNAML) of PHYLIP modified for the Apple Macintosh computer (Version 3.2, J. Felsenstein, Department of Genetics, University of Wash- ington, Seattle). The maximum likelihood method at- tempts to identify the evolutionary tree which has the highest probability of evolving the observed sequence data (Felsenstein 1981). As options in the program, we used the empirically determined frequencies of nucleo- tides and an average transition/ transversion ratio determined by painvise comparisons of all taxa. Furthermore, we tried global rearrangements as well as the jumble option in order to increase the probability that the tree with the greatest likelihood was discovered.

Microsatellite analysis Variation in nine GT(n) microsatellite loci, identified from a domestic dog genomic library and known to be polymorphic in wolf-like canids, were surveyed in Ethiopian wolves (Ostrander et nl. 1993). Detection of simple sequence alleles from genomic DNA was achieved by end- labelling one primer of the pair by a standard [lpzl'] dATP (Amersham) and T, polynucleotide kinase reaction (Sambrook et al. 1989) and performing 28 cycles of PCR amplification in a 25-pL reaction volume using 50 ng of target DNA, 2 m~ MgCl,, and 0.8 units Tn9 DNA polymerase (Promega). Two microlitres of each product was then mixed with 2 pL of formamide loading dye, and heated to 95 "C for 5 min before being fractionated on a 6% acrylamide gel containing 50% w/v urea. An M13 control sequencing reaction was run adjacent to the samples providing an absolute size marker for the simple sequence alleles. Gels were autoradiographed overnight with one intensifying screen at -70 "C.

Measures of genetic variability of the nine loci were calculated by the siosus-1 program (Swofford & Selander 1981). Genetic polymorphism for each population was measured as the mean number of alleles per locus (A), observed heterozygosity (H,) and heterozygosity expected from Hardy-Weinberg assumptions (HE) (Nei

MOLECULAR GENETICS OF THE MOST ENDANGERED C A N l D 305

M

- 97

1978, 1987). The two measures of heterozygosity are highly correlated and we focus our discussion on HE because it is considered a better index of genetic variability (Nei & Roychoudhury 1974). Deviations from Hardy-Weinberg equilibrium were tested using chi- square tests with and without pooling (Hart1 & Clarke 1988). Both tests were used because some loci had many rare alleles. For loci with pooled genotypes, genotypes were grouped into three classes (homozygotes for the most common allele, common/rare heterozygotes and other genotypes). The standardized variance in allele frequencies among populations, Fs, (Wright 1969), was calculated for single and multiple allele cases using modifi- cations described in Nei (1977) and Nei & Chesser (1983). An estimate of migration rate was obtained from the relation Fst = 1/1 + 4Nm, in which N is the population size and m is the migration rate (Slatkin 1987). Nei’s standard genetic distance and its standard error were calculated using the program DISPAN (Ohta 1993).

COYOTE

EIHIOPIAN

Results

63 -.-

Relationships of the Ethiopian wolf

The Ethiopian wolf is most similar in 2001 bp of protein coding sequence to coyotes and gray wolves, having mean ~t SD divergence values of about 5 2 0.52% (Ta- ble 2). The sequence divergence between Ethiopian wolves and other African wolf-like canids ranges from 6 k 0.57 to 11 f 0.82%. Phylogenetic trees based on the sequence data have Ethiopian wolves clustered with coyotes and gray wolves in 84% of bootstrap replications (Fig. 2). The golden jackal is a sister taxon to the clade containing these three species in 97% of bootstrap replications. A tree with golden jackals and the Ethiopian wolf as a monophyletic clade requires an additional 11 steps. Black-backed jackals appear as the sister taxon to the clade containing golden jackals, wolves, coyotes and

BIACK-BACKED JACKAL-]

W - BUM-BACKED JACKAL-2

G U Y FOX

GRAY WOLF r

SIDE-SmED IACKAL

AFRICAN WILD DOC

Fig. 2 The strict consensus tree of the two most parsimonious trees based on phylogenetic analysis of 2001 base pairs of the rntDNA sequence in wolf-like canids. Two individuals were sequenced for each species. The gray fox sequence was used to root the tree. Nodes supported in over 50”h of 1000 bootstrap rep- lication trees are indicated. Tree length = 868, consistency index (CI) = 0.66, CI excluding uninformative characters = 0.51. The sequence data contains a significant phylogenetic signal as indicated by a g l statistic of -0.56 (P < 0.02; Hillis & Huelsenbeck 1992).

Ethiopian wolves in 92% of bootstrap replicates but the branching order of wild dogs and side-striped jackals is less well resolved. The maximum likelihood tree is identical in topology to the parsimony tree except that side-

Table 2 Divergence values according to the Jukes-Cantor model (above diagonal) and number of substitution differences (below diagonal) between wolf-like canids based on 2001 bp of mtDNA sequence

Black- Black- Side- Gray Gray Ethiopian Wild backed backed striped Golden fox wolf Coyote wolves dog jackal-1 jackal-2 jackal jackal

Gray fox Gray wolf Coyote Ethiopian wolves African wild dog Black-backed jackal-1 Black-backed jackal-2 Side striped jackal Golden jackal

- 299 295 299 313 296 287 278 289

0.168 0.166 - 0.046

89 - 104 97 211 204 162 159 170 155 187 179 97 106

0.168 0.054 0.050 -

207 171 173 176 115

0.178 0.115 0.111 0.112

217 212 205 205

0.166 0.086 0.084 0.091 0.118 -

109 174 158

0.161 0.090 0.082 0.092 0.115 0.057 -

188 160

0.156 0.101 0.096 0.094 0.111 0.093 0.101

162

0.163 0.051 0.056 0.060 0.111 0.084 0.08s 0.086

306 D. G O T T E L L I et al.

Fig. 3 Example autoradiogram of SstI mtDNA restriction fragment patterns for 11 Ethiopian wolves and two sympatric domes- ticdogs. -

striped and African wild dog nodes are interchanged. All branch lengths are significantly different from zero (P~0.01) . In summary, phylogenetic analysis of 2001 base pairs of mitochondrial sequence data suggests that the closest living relatives of the Ethiopian wolf are gray

Fig. 4 Example of an autoradiograph of the dinucleotide microsatellite repeat locus 377 amplified in Ethiopian wolves. Allele sizes in base pairs @p) are indicated, the domestic dog allele (0) is the 130-bp repeat. Genotype designations shown above lanes.

and Ethiopian wolves were substantially different. All eight Ethiopian wolves sequenced and Di, had identical control region sequence which differed from the two domestic dogs by 11%. These results coupled with the obser- vation that all of the Ethiopian wolves in the Web Valley had identical mtDNA genotypes to those from the Sanetti plateau, an area without resident domestic dogs, suggests that hybridization between female domestic dogs and male Ethiopian wolves is rare or absent.

wolves and coyotes rather than other jackals or the Afri- can wild dog.

Micrmatellite variation

We found no-mtDNA restriction site polymorphism in our sample of 23 Ethiopian wolves from Sanetti and Web Valley (eg. Fig. 3). We observed 69 restriction fragments using 17 restriction enzymes and consequently sampled variation in an estimated 412 bp. In addition, we found no variation in 394 bp of control region sequence among the eight individuals representing four different social groups in the two Ethiopian wolf populations. Appar- ently, the Ethiopian wolves we have sampled belong to a single mitochondrial lineage of very recent common ancestry.

We compared restriction fragment patterns of Ethio- pian wolves with those found in Seven sympatric domestic dogs, two phenotypically abnormal Ethiopian wolves and Di, an individual suspected to be a Ethiopian wolf- dog hybrid. We also compared Ethiopian wolf restriction fragmentpatterns to those found previously in a panel of 32 dog breeds (Table 1, Wayne et al. 1992). All normal Ethiopian wolves and abnormal individuals analysed had identical restriction fragment patterns. These differed from the pattern identified in dogs for 14 of the 17 restriction enzymes used. Dogs and Ethiopian wolves share, on average, 5642% of restriction fragments, corresponding to an estimated sequence divergence of approximately 3.5% (Nei & Li 1979). Control region sequences of dogs

The nine microsatellite loci that we surveyed were mod- erately polymorphic in Ethiopian wolves (e.g. Fig. 4). The number of alleles varied from one to five and levels of heterozygosity ranged from 0 to 0.534 (Table 3, Fig. 5). Genotype distributions for each locus fit Hardy- Weinberg expectations as determined by chi-square tests (P < 0.05). Overall heterozygosity and allelic diversity ap- peared lower in the Sanetti population; only six of nine loci were polymorphic and average levels of heterozygosity were significantly lower (arcsine-transformed t-test, P > 0.05; Archie 1985). However, allele frequencies differed little between the two populations and the overall Fs,, 0.057, was small (Fig. 5). This F,, value primarily re- flects differing frequencies of the M and P allele a t locus 213 (Fig. 5). Migration rates based on this value of F, are large, about 4.3 migrations per generation. More than one migrant per generation is theoretically sufficient to con- found genetic divergence due to drift in finite populations (Slatkin 1987). The Nei’s standard genetic distance between the two populations is only 0.022 * 0.02. In con- trast, the standard genetic distance between domestic dogs and Web and Sanetti Ethiopian wolves is 1.73 L 0.48 and 1.91 2 0.54, respectively.

In comparison to other wolf-like canids, Ethiopian wolves have low levels of allelic diversity and heterozy-


Table 3 Number of alleles and heterozygosity of the nine microsatellite loci surveyed in two Ethiopian wolf populations and in domestic dogs. Standard errors in parentheses

Sample No.of H, Locus Population size alleles He

225

109

204

123

377

250

213

173

344

Mean

web valley 22 Sanetti 15 domestic dogs 35 web valley 23 Sanetti 15 domestic dogs 36 web valley 24 Sanetti 15 domestic dogs 35 web valley 24 Sanetti 16 domestic dogs 40 web valley 23 Sanetti 18 domestic dogs 40

Sanetti 15 domestic dogs 40




web valley 22

web valley 22

web valley 22

web valley 22

web valley 22.8 (0.3)

(0.4) Sanetti 16.4

Domestic dogs 35 (5.3)

2 2 4 4 2 5 2 1 5 3 1 5 3 5 9 3 2 10 2 2 9 3 2 5 3 1 6

2.8

2.0

6.4 (0.8)

(0.2)

(0.4)

0.206 0.129 0.569 0.503 0.231 0.700 0.042 0.000 0.564 0.121 0.000 0.802 0.165 0.203 0.678 0.280 0.129 0.828 0.206 0.063 0.569 0.534 0.497 0.763 0.382 0.000 0.636

0.355 (0.077) 0.201

(0.075) 0.570

(0.081

0.227 0.133 0.514 0.565 0.267 0.500 0.042 0.000 0.333 0.125 0.000 0.286 0.174 0.222 0.476 0.318 0.133 0.500 0.227 0.063 0.514 0.682 0.533 0.600 0.455 0.000 0.923

0.304 (0.061) 0.179

(0.066) 0.729

(0.045)

gosity. The allelic diversity and heterozygosity of other closely related canids varies from 4.5 1.1 to 6.4 0.8 and 0.520 k 0.103 to 0.729 k 0.045, respectively (Table 4; Roy et al. in press). The mean value of allelic diversity and heterozygosity of six wolf-like canids was 5.2 f 0.54 and 0.628 f 0.08, respectively. Therefore, Ethiopian wolves have 38 and 46% of the average allelic diversity and heterozygosity of six species of wolf-like canids.

Although domestic dogs and Ethiopian wolves only differ by about 3.5% in mitochondria1 DNA sequence (see above), the two species differ considerably in microsatellite allele frequencies. Of the 19 alleles found in phenotypically normal Ethiopian wolves, eight (42%) are unique to them (Fig. 5) . Similarly, of 57 alleles in dogs, 47 (82%) are not found in phenotypically normal Ethiopian wolves. Consequently, it is possible to define a set of marker alleles to determine if Ethiopian wolves that are phenotypically aberrant have alleles otherwise found only in domestic dogs. Genotypes from all nine loci show that the seven phenotypically abnormal Ethiopian wolves possess alleles not found in normal Ethiopian wolves, but which are present in domestic dogs (Fig. 5). Individuals 53, S5, W7 and W9 have alleles at two loci (S3: 377-0,173-

and individuals W5, W8, and W11 alleles at one locus (W5: 109-1; W8,Wll: 344-B) that are otherwise only found in dogs (Fig. 5). No other Ethiopian wolves have alleles found in dogs that are not also found in the Sanetti Ethio- pian wolves, an area free of domestic dogs. The presence of these alleles in the Ethiopian wolves identified as being phenotypically abnormal suggests hybridization between dogs and Ethiopian wolves has occurred. How- ever, the presence of dog alleles is not restricted to Web Valley; four phenotypically normal individuals from Sanetti (83, N19, N17 and N2) also have one allele at locus 377 (K or M, Fig. 5) that otherwise is found only in domestic dogs.

A; S5: 109-F, 204-G; W 7 109-F, 250-H; W9: 123-G, 344-B),

Table 4 Mean sample size, allelic diversity (mean number of alleles per locus) and mean heterozygosity for nine microsatellite loci surveyed in wolf-like canid populations (Roy et al. in press). Standard error among loci averaged over all populations in parentheses

Population Details

Mean heterozygosity Sample size Allelic Hard y-Weinberg per Locus diversity expected

Gray wolf nonhybridizing 17.7 (2.8)

Coyote 6 populations; Alaska to Maine 17.0 (3.0) Red wolf captive colony 29.9 (1.0) Golden jackal Kenya 16.4 (0.7) Black-backed jackal Kenya 13.7 (1.7) Ethiopian wolf Ethiopia; 2 populations 19.6 (0.4) Domestic dogs 32 breeds 26.0 (5.3)

5 populations; Alaska to Quebec 4.5 (1.1)

5.3 (0.8) 5.9 (0.73)

4.8 (0.8) 5.0 (0.4) 2.4 (0.3) 6.4 (0.8)

0.620 (0.070) 0.675 (0.035) 0.548 (0.072) 0.520 (0.103) 0.674 (0.063) 0.241 (0.063) 0.729 (0.045)


1.0- LOCUS 225 1.0 1 LOCUS 377

0.8

0.6

0.4

0.2

0.0

B I J K L M N O P Q A B C E F

LOCUS 344 LOCUS 250

n, ~,l,~ 0.2

0.0 B D E F

1.0 -) LOCUS 109

Fig. 5 Allele frequency histograms for nine microsatellite loci in Ethiopian wolves from the Web Valley and Sanetti Plateau and domestic dogs. Consecutive letters differ by a single two base pair repeat unit. See Ostrander et al. (1993) for further details on each locus.

Known pack affiliations and relationships of the suspected dog-Ethiopian wolf hybrids suggest the presence of dog alleles is occasionally due to recent common ancestry. Three individuals with dog alleles [W8, W9 and Wll], are litter mates from a known mother, W6 (Ta- ble 1). Individual W10 is also part of this litter but does not display dog alleles nor is it phenotypically abnormal. Consequently, this litter may have had two fathers, one being an Ethiopian wolf and the other, a domestic dog or dog-Ethiopian-wolf hybrid. However, a single male genotype can be constructed that would satisfy the observed segregation. More definitive evidence for multiple paternity in Ethiopian wolves is provided by analysis of a litter from female W2. Her three offspring show four alleles (F, G, H, I) at locus 109 for which she is a homozygote for the G allele. Therefore, assuming a germ line mutation has not occurred, the litter must have had two fathers. The occurrence of a dog allele at locus 109 for individual W7 may be explained if Male S5 from a neighbouring pack is his father. Male S5 is the only individual that cannot be excluded as the father of W7 among the sampled wolves and has the same dog allele at locus 109 (Fig. 5) .

The earlier mitochondrial DNA analysis suggested that interbreeding of female domestic dogs and male Ethiopian wolves is not common. This conclusion is con- sistent with field observations and questionnaire results indicating that female dogs do not attempt mating with male Ethiopian wolves (Sillero-Zubiri & Gottelli 1991; Gottelli & Sillero-Zubiri 1992). However, only three of the seven phenotypically abnormal individuals had been analysed for mitochondrial DNA variation. Conse- quently, we analysed the four other phenotypically abnormal individuals for mitochondrial DNA restriction fragment polymorphisms unique to domestic dogs. Us- ing four restriction enzymes that produced diagnostic dog or Ethiopian wolf profiles, we found that all of the four individuals had Ethiopian wolf genotypes. We were also able to use a minute quantity of DNA remaining from the Di sample to amplify microsatellite locus 204, at which her genotype was CG indicating she was a heterozygote for a dog allele (Fig. 5). Therefore, we con- clude that the seven phenotypically abnormal Ethiopian wolves and Di, a satellite female with an unusual pheno- type, have an ancestry involving hybridization between male domestic dogs and female Ethiopian wolves.


Discussion

Phylogenctic relationships of Ethiopian wolves

The phylogenetic analysis of 2001 bp of protein coding sequence suggests that the closest living relatives of Ethiopian wolves are not other African wolf-like canids, but gray wolves and coyotes. We hypothesize that Ethio- pian wolves may be an evolutionary relic of a past invasion of Northern Africa by gray wolf-like ancestors. Fos- sils of gray wolf-like canids are known from Europe and Asia from the Late Pleistocene (Kurt& 1968). The gray wolf-like progenitors of Ethiopian wolves may have been preadapted to existence in the cold-temperate Ethiopian highlands given that gray wolves flourish in temperate and alpine conditions and occupy a vast geographic range including much of the Northern Hemisphere. The gray wolf-like progenitors of Ethiopian wolves may not have advanced farther south into the East African savannah because of competition with the more specialized, tropically adapted African wild dog Lycaon pictus. Inter- specific competition among large savannah-adapted carnivores is intense and may lead to the exclusion of less well adapted forms (see discussion in Van Valkenburgh 1988). Because the Ethiopian wolf has a wolf-like pheno- type and does not have a direct ancestor-descendant rela- tionship with African jackals we have used this common name rather than the more frequently used Simien jackal (Sillero-Zubiri & Gottelli, in press).

The lack of mitochondrial DNA variability in Ethio- pian wolves contrasts with other species of wolf-like canid (Table 5 ) . Even the endangered East African wild dog, having a population size of fewer than several thousand individuals, shows at least three distinct restriction fragment genotypes (Ginsberg & Macdonald 1990; Girman et al. 1993). In a nonsubdivided, freely interbreeding population at equilibrium, the effective number of &DNA restriction fragment genotypes can be esti-

mated as ne = 2 N y + 1 where N, is the effective female population size and p is the mutation rate (Birky etal. 1983; Birky et al. 1989; Lehman & Wayne 1991). Assuming a liberal estimate of N, = 150 and given our sample of 412 base pairs, p = 1.1 x restriction-site substitutions per restriction site genome per generation (Lehman & Wayne 1991) and ne = 1.003. Therefore, the presence of only one mtDNA genotype in Ethiopian wolves from the Bale Mountain National Park is expected given the presence of a small effective population size and equilibrium conditions. Coalescence to a single genotype might be expected after 4Nf generations (Avise et al. 1988) or approximately several hundred years.

For the nine microsatellite loci, levels of allelic diversity and heterozygosity were also substantially lower than found in other wolf-like canids (Table 4; Roy ef al., in press). The level of allelic diversity and mean heterozygosity was roughly 40% of that of other wolf-like canids. In a stepwise mutation model, as might apply for microsatellite loci (Valdes et al. 1993), and for populations in drift-mutation equilibrium, H = 1 - [ l / ( l + 8Ny)05], where His the expected heterozygosity and p is the mutation rate (Ohta & Kimura 1973). Assuming a mutation rate of about lo-' for microsatellite loci (Dietrich etal. 1992; Dallas 1992), the expected population size for the observed average heterozygosity value of 0.24 is about 914 individuals. If an equal proportion of males and females reproduce, then about half this value is the female effective population size. According to the previous equation relating mitochondrial variability to population size, an equilibrium female population of this size would be expected to have approximately only one mtDNA genotype.

In conclusion, both microsatellite and mitochondrial DNA analysis suggest small population sizes may have characterized the recent evolution of Ethiopian wolves. Population census reports show that Ethiopian wolf popuIations have been declining due to loss of habitat

Table 5 Number of mtDNA restriction site genotypes, maximum percentage mtDNA sequence divergence within a species, sample size (number of individuals) and maximum distance between sampling localities for ten canid species

Number of YO Sequence Sample Max. Distance Species genotypes divergence size between sites (km) Reference

Ethiopian wolf Canis simensis Black-backed jackal Canis mesomelas Golden jackal Canis nureus Side-striped jackal Canis adustus Coyote Conis lotrans Gray wolf Cunis lupus Kit fox Vulpes macrotis Red fox Vulpes vulpes Channel Island fox Urocyon littoralis African wild dog Lycuon pictrrs

1 4 2 2

32 9

24 3 5 6

0.0 8.4 0.1 0.2 2.5

1.5 1 .z 1.8 0.9

0.8

54 64 20 7

327 276 256

4 150 104

20 400 400 400

4800 15000 2000

12000 250

3000

This article Wayne et nl. (1990) Wayne et nl. (1990) Wayne et nl. (1990) Lehman & Wayne (1991) Wayne ef ul. (1992) Mercure et nl(1993) Geffen et al. (1992) Wayne et nl. (1991) Girman et nl. (1993)


and human activities at least since the last century (Morris & Malcolm 1977; Yalden & Largen 1992). Even over the long term, Ethiopian wolf population sizes may have been progressively decreasing. The Afro-alpine habitat of Etluopian wolves was geographically much more extensive during the last glacial interval (10 000-70 000 years ago) when the African tropics were generally cooler and drier than today (Kingdon 1990; Yalden 1983; Bonnefille ef al. 1990). Fifteen thousand years ago, the afro-alpine habitat zone of Ethiopia extended about 1000 m lower than at present (Flenley 1979) and would have occupied about 100 000 km2. Today, a mere 2% of this area (2275 km2) is Afro-alpine habitat (Gottelli & Sillero Zubiri 1992). Considering this immense decrease in suitable habitat, the population sizes of Ethiopian wolves have probably been declining rapidly over the past 15000 years. Only recently has this decline been further acceler- ated by the activities of humans. Both processes might have led to the reduced levels of variability found pres- ently in Ethiopian wolves.

Hybridization also threatens the genetic integrity of Ethiopian wolves. Microsatellite analysis suggests that the seven individuals identified as phenotypically abnormal all show evidence of hybridization with dogs. These individuals were found to be active members of Ethio- pian wolf packs and at least one individual may have produced viable offspring. Phenotypically abnormal individuals represent about 17% of the sampled population implying hybridization is having a substantial effect on the genetic character of Ethiopian wolves. In support of this conclusion, a field survey of 156 individuals from the two localities found none in Sanetti (n = 67) and 12 of 89 (13.5%) in Web Valley, that had abnormally coloured pelage (Gottelli & Sillero-Zubiri 1990; Sillero-Zubiri & Gottelli 1991; Gottelli & Sillero-Zubiri 1992). The presence of dog alleles in Sanetti Ethiopian wolves, an area without domestic dogs, suggests gene flow from Web Valley may have caused the inkogression of dog alleles into that population or may represent rare variants that have ap- peared through mutation. The value of Fs, between Sanetti and Web Valley populations indicates genetic exchange between them is occurring or has occurred in the recent past and is sufficient to counteract differentiation due to genetic drift.

Interspecific hybridization apparently occurs between male domestic dogs and female Ethiopian wolves and might happen even if a receptive female has also mated with an Ethiopian wolf. The actual number of interspecific hybridization events is difficult to recon- struct because the sampled individuals with dog alleles may be several generations removed from the original interspecific cross. The presence of domestic dogs in the Web Valley poses genetic, ecological and disease threats to Ethiopian wolves (Mebatsion et al. 1992; Macdonald, in

press). Although the control of domestic dogs in the Bale Mountains National Park has been proposed (Gottelli & Sillero-Zubiri 1990, 1992), it has not been seriously considered, in part, because of lack of genetic evidence for hybridization. This study provides convincing evidence that hybridization with dogs has occurred in Ethiopian wolf populations.

In summary, we have shown that the Ethiopian wolf is a phylogenetically distinct canid, related to the gray wolf and coyote, that may be a relic form remaining from a more extensive Late Pleistocene invasion of the Ethiopian highlands by wolf-like progenitors. Ethiopian wolves have lower mitochondrial and microsatellite variability than other large canids, probably reflecting population decline due to habitat reduction since the end of the Pleistocene and, more recently, to human activities. Ethiopian wolves are threatened by the presence of domestic dogs who hybridize with them, compete for food and transmit diseases. We recommend that the feral domestic dogs be controlled to eliminate these threats and that a programme of captive breeding be initiated imme- diately with genetically pure founders. Demographic and genetic surveys of other smaller populations on both sides of the Rift Valley are also needed.

Acknowledgements

We would like to thank E. Geffen, E. Barratt, 8. Van Vakenburgh, M. Bruford and three anonymous review- ers for helpful comments and assistance with the analysis. We gratefully acknowledge G. Amato and C. Melleresh for their help with sample disposition and laboratory techniques. Field research on the Ethiopian wolf was funded by the New York Zoological Society (The Conservation Society) and was performed under the auspices of the Ethiopian Wildlife Conservation Organi- zation. This study was partially funded by the Institute of Zoology and the Consultancy and Conservation Division both of the Zoological Society of London, The People’s Trust for Endangered Species and a National Science Foundation Grant to R.K.W. (BSR 9020282) and a USPHS National Research Service Award (GM-07104) to D.J.G.

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Documents

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis