Mitochondrial DNA Sequence Variation in Portuguese Native Dog Breeds: Diversity and Phylogenetic Affinities

Journal of Heredity 2006:97(4):318–330 ª The American Genetic Association. 2006. All rights reserved. doi:10.1093/jhered/esl006 For permissions, please email: [email protected]. Advance Access publication July 3, 2006 Mitochondrial DNA Sequence Variation in Portuguese Native Dog Breeds: Diversity and Phylogenetic Affinities

ANA ELISABETE PIRES,LAHOUSSINE OURAGH,MOHSEN KALBOUSSI,JOSE´ MATOS, FRANCISCO PETRUCCI-FONSECA, AND MICHAEL W. BRUFORD

From Centro de Biologia Ambiental, Faculdade de Cieˆncias da Universidade de Lisboa, Rua Ernesto Vasconcelos, Edifıćio C2-3 Piso, 1749-016 Campo Grande, Portugal (Pires and Petrucci-Fonseca); Instituto Nacional de Engenharia, Tecnologia e Inovacxaõ, Estrada do Pacxo ao Lumiar, 22, Edifıćio E, Molecular Biology Group, 1649-038 Lisboa, Portugal (Pires and Matos); Laboratoire d’Analyses Geńe´tiques Ve´te´rinaires, Institut Agronomique et Ve´te´rinaire Hassan II, BP 6202-Instituts, 10101-Rabat, Morocco (Ouragh); Department of Biology, Faculty of Sciences of Tunis, University El Manar, Tunisia (Kalboussi); and Cardiff University, School of Biosciences, P.O. Box 915, CF10 3TL, Cardiff, United Kingdom (Pires and Bruford).

Address correspondence to A. E. Pires at the address above, or e-mail: [email protected].

Abstract In an extensive survey of the genetic diversity in Portuguese dogs, we have examined an 887-bp fragment of the mitochondrial DNA (mtDNA) from 8 Portuguese, 1 Spanish, and 2 North African native dog breeds, including village dogs from Portugal and Tunisia. Forty-nine haplotypes were found in the 164 individuals analyzed, with private haplotypes being found in several breeds. For example, the Castro Laboreiro Watchdog, a rare breed from a small and isolated region in Portugal, was mono- morphic for mtDNA and possessed a new haplotype, which may be provisionally considered a breed-specific marker. Phy- logenetic analyses recapitulated 4 major clades identified in other studies, but new haplotypes, grouping within a clade that was previously thought as geographically restricted, were detected in Portugal and Morocco. Portuguese village dogs showed no genetic differentiation from nonnative dogs or from local breeds of the areas in which the village dogs were sampled. Although Iberian and North African dog breeds possessed breed-specific mtDNA haplotypes, no significant geographic structure could be detected among them. There is no evidence for introgression of North African haplotypes in Iberian dogs, contrary to previous results for other domestic animals.

It is now well established that the ancestor of the dog, Canis 400 dog breeds worldwide, and their economic importance is familiaris, is the gray wolf Canis lupus. Gray wolves and dogs substantial (Clutton-Brock 1984). differ by only 4.6% (12 substitutions in a 261-bp fragment of Although dogs were present in what is now Portugal by the D-loop) in their mitochondrial DNA (mtDNA) (Vila` and the Mesolithic (Cardoso 2002), the first breeds (probably others 1997), and this comparison is the lowest value be- used for hunting) are known from mosaic depictions dating tween any pair of canid species. Other lines of evidence also from the Roman occupation (Braga 2000), and it is probable place the wolf as the dog ancestor, such as morphology that the Phoenicians had previously brought hound-type (Wayne 1986), behavior (Zimen 1981), and vocalization dogs into the country (Veiga 2001). Currently, there are 10 (Lorenz 1975; Zimen 1981). working native dog breeds, recognized by the Portuguese Currently, the domestic dog is the most abundant canid, Kennel Club, that as a group demonstrate remarkable phe- with a global population of around 400 million animals, and notypic diversity. Seven of these dog breeds are also recog- has the widest geographic distribution of any domestic spe- nized by the Fe´de´ration Cynologique Internationale. These cies (Coppinger R and Coppinger L 2002). Among mammals, breeds are thought to have a relatively recent origin although domestic dogs are remarkable by the range of natural varia- their foundation dates are unknown. Breed standards date tion they exhibit in terms of morphological and behavioral mostly from the first half of the 20th century, and intensive traits, even if the maintenance of dog breeds is guided by selection for standards started only 20–30 years ago. established standards, such that within each breed animals Recent demographic changes are predicted to have im- often show an uniform and distinctive morphology and be- pacted the genetic diversity of Portuguese breeds. For exam- havior (Ostrander and Comstock 2004). There are more than ple, in the past, transhumance (twice-yearly migration of

318 Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs livestock, shepherds, and their various livestock guarding dog The most recent phylogenetic analysis of mtDNA in do- breeds [Martı´n and others 1994]), which has not occurred mestic dogs, including samples from Europe, Asia, Africa, in Portugal since the 1990s (Silva 2000) and in Spain since and Arctic America, assigned mtDNA sequences to 6 clades: the 1950s (Coppinger R and Coppinger L 2002), is likely A, B, C, D, E, and F (Savolainen and others 2002). At least to have facilitated gene flow among livestock guarding 5 wolf matrilines are considered to be present at the origin of dog populations and breeds when they were brought into the domestic dog population because all clades but F are contact during these seasonal migrations. Further, some Por- intermingled with wolf sequences. Clades A, B, and C contain tuguese dog breeds have gone through periods of critically 95.9% of the sampled dog haplotypes and are represented low population size (Vasconcelos 1995; Gomes 2003) due in all geographic regions (clade A) or in all except America to rural emigration, the abandonment of agricultural and (clades B and C). Geographically more restricted are the hunting practices, and major reductions of wolf populations clades D, E, and F, which, respectively, are found in Turkey, in some regions ICN (1997). With fewer wolves in the wild, Spain, and Scandinavia; Japan and Korea; and Japan and Siberia. shepherds began using dogs with no specific skills for live- This is the first comprehensive study of mtDNA varia- stock guarding, and the subsequent decrease in the use of tion of Portuguese dogs and some other geographically ad- livestock guarding dog caused a reduction in their number. jacent populations—the Spanish mastiff, Aidi, Sloughi, and Finally, the popularity of dog shows created subsets of de- Tunisian mongrel. Table 1 summarizes the function, country mographically isolated dogs within some breeds (i.e., show vs. of origin, and conservation status of these breeds. The major working dogs). Portuguese livestock guarding dogs are a aim of this investigation was to determine the mtDNA ge- good example of this, where both subsets have become dis- netic variability of Portuguese dogs and to evaluate the geo- tinct, not only morphologically (C. Cruz, personal commu- graphic context of genetic variation in these and neighboring nication) but also genetically (Petrucci-Fonseca and others dog populations. 2000). Most of the demographic data collected from the Por- tuguese Kennel Club refers only to show dogs, and therefore, the number of registrations per year does not necessarily re- Methods flect the total population at any given time. Almost all breeds Sampling and DNA Extraction show a common pattern, concerning the evolution of their numbers, of an increase in the number of registries in the Blood, hair, and tissue samples from 164 animals belonging Portuguese Kennel Club by the end of the 1970s (Gomes to 13 different breeds or populations of dogs were collected 2003). at dog shows, from breeding kennels, and from distinct loca- Spain and the Maghreb region (Morocco, Tunisia, and tions in their historical breed regions (Table 1). Animals were Algeria) are geographically proximate to Portugal and are selected based on morphological standards and on informa- all within the same zoogeographic region: the Mediterranean tion about their ancestry in order to exclude related animals subregion of the Palaeartic (Roger and others 1998). Histor- back to the third generation whenever possible, although ically, the Iberian Peninsula has had a close connection with complete information on ancestry was not always available. North Africa mainly due to the Moorish occupation, which In the Portuguese Warren hound, 3 body size types occur: lasted for 8 centuries (Brito and others 1992; Ribeiro and small, medium, and large. Hence, individuals from this breed Saraiva 2004). The Moors introduced crops and livestock were sampled more extensively. Since the 1960s, one Portu- to Iberia (Cymbron and others 1999), and it is possible that guese Water dog breeder has exported dogs to outside of livestock guarding dogs were traded as well. In order to assess Portugal, mainly to the United States (Molinari 1993), and genetic variation between domestic dog populations from one animal from the United States is included, possibly rep- North Africa and Iberia, the 2 internationally recognized resenting an extinct lineage in Portugal. Samples were also North African dog breeds, the Aidi and Sloughi, and mongrel collected from dogs kept in municipal kennels or animal shel- dogs from Tunisia were included in our study. ters, whose phenotypes could not be assigned to any recog- MtDNA is a powerful tool for estimating levels of genetic nizable breed (hereafter Portuguese village dogs) as defined diversity, phylogenetic structure, and recent demographic by McDonald and Carr (1995). We selected those individuals history in domestic animals, but its use beyond these appli- from diverse regions such as the Azores, where the Azores cations is more limited (Bruford and others 2003), especially cattle dog originated, the Estrela Mountain region, where the because it only provides information about the female lineage Estrela Mountain dog originated, and Alentejo, where the (Avise 2004). This limitation is a drawback when studying Alentejo Shepherd dog originated. In Tunisia, there are no domestic animals because male-mediated gene flow is usually formally established dog breeds, and consequently, samples more pronounced among them. For example, the application were collected randomly within the local population. To min- of mtDNA markers in domestic dogs (e.g., Okumura and imize the risk of both the effects of genetic relatedness others 1996; Tsuda and others 1997; Vila` and others 1997; among individuals and introgression from other breeds Savolainen and others 2002) has showed little correspon- and/or village dogs, we sampled for each breed, working ani- dence between mitochondrial lineages, geographic structure, mals with unknown ancestry only when from remote regions or traditional breed classification, and many breeds contain within the historical range of each breed. haplotypes shared by other breeds scattered over different The number of registered females for each breed was col- phylogenetic clades. lected directly from Portuguese Kennel Club archives. The

319 320 Heredity of Journal 2006:97(4)

Table 1. Information on the dog breeds used in this study. LGD, livestock guarding dogs; LHD, livestock herding dogs; HD, hunting dogs; WD, water dog. Harmonic means of breeding females (2–8 years of age) were calculated taking into account the entire time span of records at the Portuguese Kennel Club for each breed. Demographic data only available for Portuguese autochthonous dog breeds

Harmonic mean Current Number of female population Current female conservation Total number Nonshared New haplotypes Breed/population of dogs samples Locality Function size (time span)a population size status of haplotypesb haplotypesb (global context)c Castro Laboreiro Watchdog 14 Portugal LGD 6 (1932–2001) 435 Endangered 1 1 1 (CLWD) Estrela Mountain dog (EMD) 15 Portugal LGD 7 (1932–2001) 3692 Vulnerable 8 3 2 Alentejo Shepherd dog (ASD) 15 Portugal LGD 12 (1933–2001) 1303 Vulnerable 8 3 0 Azores cattle dog (ACD) 14 Portugal LHD 81 (1985–2001) 1468 Vulnerable 4 0 0 Portuguese Sheepdog (PSD) 8 Portugal LHD 57 (1954–2001) 414 Endangered 2 1 0 Portuguese Pointer (PP) 9 Portugal HD 43 (1932–2001) 2167 Vulnerable 2 0 0 Portuguese Warren hound 27 Portugal HD 44 (1932–2001) 1483 Vulnerable 13 6 4 (PWH) Portuguese Water dog (PWD) 11 Portugal WD 34 (1946–2001) 932 Endangered 5 3 0 Spanish mastiff (SM) 10 Spain LGD ———621 Aidi 10 Morocco LGD ———841 Sloughi (SL) 10 Morocco HD ———652 Tunisia dogs (Tun) 9 Tunisia —— — — 620 Portuguese village dogs 12 Portugal —— — — 10 7 2 (PortVillageDogs) Total 164 37 13

a Time span refers to the time period since the first Portuguese Kennel Club records for each breed until 2001. b Total number of haplotypes and number of nonshared haplotypes detected in the Iberia and North Africa regarding the 887-bp mtDNA fragment. c New haplotypes based on the trimmed 554-bp mtDNA fragment described above. Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs number of potentially breeding females for each breed over cler, with an initial denaturation step of 94 C for 3 min; time was calculated as the harmonic mean of breeding followed by 40 cycles (60 cycles for hair samples) of dena- females since the first registries until the year 2001 (the ma- turation at 94 C (40 s), annealing at 55 C (30 s), and exten- jority of females only breed from the age of 2 up to 8 years sion at 72 C (1 min); followed by a final extension step at old) because this is the least biased stationary estimator when 72 C for 7min. For hair samples, we used a ramp between comparing fluctuating, rapidly expanding or contracting, the annealing and extension steps at 50% of the maximum populations (Frankham and others 2002). These calculations speed (programmable in the thermocycler) to make the tem- are based on the Portuguese Kennel Club records only and perature transition slower and increase the number of stable mainly involve show dogs. Currently, these are the most ac- primer–template complexes undergoing extension in each cy- curate demographic data available due to a lack of a reliable cle. A negative control was included in each set of amplifi- census of working dogs for each breed. cations. PCR products were separated on 1.5% agarose gels, Blood samples (2 ml) were taken into vacutainers contain- cleaned using the GeneClean Turbo kit (Qbiogene, Carlsbad, ing ethylenediaminetetraacetic acid (EDTA) (10% w/v) and CA), and eluted in 35 ll of elution solution. Purified products kept frozen until processed. Hairs, including the bulb, were were sequenced in both directions using the ABIPrism plucked (up to 40–50 per individual) and kept dry. Tissue BigDye Terminator Cycle Sequencing Ready Reaction Kit samples (ear biopsies) were preserved in dimethyl sulfoxide (version 2.0) and following the manufacturer’s instructions. (DMSO)–salt buffer (20% DMSO, 0.25 M Na–EDTA, and Products were separated on a semiautomated DNA analyzer NaCl to saturation, pH 8.0) at 20C. DNA was extracted (ABI 377) and sequences were edited, assembled, and aligned from whole blood and tissue according to standard protein- using the program SEQUENCHER 3.1.2 (Gene Codes ase K/Phenol–Chloroform protocols (Sambrook and Russel Corporation) and submitted to GenBank using SEQUIN 2001), followed by an extra ethanol precipitation step, or us- (http://www.ncbi.nlm.nih.gov/Sequin/). ing the Nucleospin Blood QuickPure kit (Macherey-Nagel, Du¨ren, Germany) following the supplier’s recommendations. Population and Phylogenetic Analyses DNA was extracted from hair bulbs in a Chelex 20% solution (protocol adapted from Walsh and others 1991). DNA Polymorphism Arlequin version 2.0 (Schneider and others 2000) was used to compute indices of genetic diversity. Haplotype diversity H DNA Amplification, Purification, and Sequencing and nucleotide diversity p were determined for each popula- An mtDNA fragment of 887 bp, comprising a segment of the tion. Pairwise genetic distances between haplotypes were cal- cytochrome b, the tRNA-Thr, the tRNA-Pro, and a segment culated under the Tamura–Nei model (Tamura and Nei 1993), of the control region, was amplified using a single pair of after excluding positions with insertion/deletions. Heteroge- primers (all primers are numbered in relation to the complete neity of substitution rates per site across the D-loop region mtDNA sequence determined by Kim and others 1998): was taken into account and gamma distribution parameters L15210 5#-ACA TGA ATT GGA GGA CAA CCA GT-3# (Yang 1994) estimated with the best-fitting model of sequence (a shortened version of Shields and Kocher’s [1991] evolution, as determined by MODELTEST version 3.06 # L15774 primer) and H16097 5 -TAT GTC CTG TGA (Posada and Crandall 1998). Pairwise FST values were estimated CCA TTG ACT GA-3# (S. Funk, Institute of Zoology, Lon- and significance at the 5% level determined with 10 000 per- don). L15210 plus the following 5 internal primers were used mutations. Arlequin was also used to carry out an analysis of for sequencing: H15377 5#-TTT GAG TCT TAG GGA the molecular variance (AMOVA; Excoffier and others 1992). GGG CG-3# (designed by A. E. Pires), L15360 (Hoelzel Three groups were defined taking into account the geographic and others 1991), L15515 5#-GTG TCA GTA TYT CCA origin of the samples: Portugal, Spain, and North Africa. GGT-3# (designed by A. E. Pires), L15805 (Southern and Sequences with no missing data were collapsed into hap- others 1988), and H16075 5#-GCA CCT TGA TYT TAT lotypes using the program COLLAPSE (Version 1.0), avail- GCG T-3# (designed by A. E. Pires). able at http://darwin.uvigo.es. All haplotypes were compared Polymerase chain reaction (PCR) amplifications were with the DNA sequence information stored in the GenBank performed in a 25-ll reaction volume with 100–200 ng of database. This was conducted using the ‘‘basic local alignment DNA from blood and tissue samples or 8–10 ll of extract search tool’’ (BLAST) program (Altschul and others 1990), from hair bulbs, as template, 1 U of Taq polymerase (Gibco available through the National Center for Biotechnology [now Invitrogen], Paisley, UK), 1 reaction buffer (Gibco Information (Bethesda, MD). Chiperm version 1.2 (Posada 10 stock contains 200 mM Tris–HCl pH 8.4 and 500 mM 2000) was used to test for significant associations between KCl), 1.5–2 mM MgCl2 (Invitrogen, Paisley, UK), 20 lMof haplotypes and breeds or dog populations using the algorithm each deoxynucleoside triphosphate (ABgene, Epsom, UK), developed by Hudson and others (1992). The significance of 0.5 lM of each primer, and Sigma water. Nonacetylated bovine the v2 statistics was approximated by Monte Carlo simulation serum albumin (MBIFermentas, Vilnius, Lithuania) was in- (Roff and Bentzen 1989) by permuting the contingency cluded at a concentration of 0.024 lg/ll for amplification tables; 100 000 permutations were performed. of blood-extracted DNA and 0.48 lg/ll for hair bulbs- Phylogenetic relationships among haplotypes (887 bp) extracted samples. Amplification reactions were performed were estimated using the neighbor-joining (NJ) method in in a GeneAmp PCR System 9700 (Perkin Elmer) thermal cy- PAUP version 4.0b10 (Swofford 2002). In order to analyze

321 Journal of Heredity 2006:97(4) the sequences reported in this study in a wider context, 54 fragment), and, of these, only positions 4, 56, 145, 162, 206, haplotypes of 554 bp (accession numbers: AF531664, and 225 are outside the control region. Changes corre- AF531668, D83609, AF531670, AF531671, AF531672, sponded to synonymous transitions. A strong transitional AF531674, AF531675, D83606, AF531679, AF531682, bias of 39.2:1 was observed, a common finding in the AF531685, AF531687, AF531656, AF531693, AF531696, mtDNA of mammals (Graur and Li 2000). Due to indels, AF531700, AF531702, AF531710, AF531658, AF531731, 3 sites contained gaps (positions 255, 722, and 729). Nucleotide AF531732, AF531733, AF531734, D83607, D83625, frequencies for the entire fragment were A 5 0.28420, D83634, AB007402, AF531722, AF531723, AF531724, C 5 0.26180, G 5 0.15250, T 5 0.30150. The low guanine AF531725, AB007380, D83601, AF531728, AF531729, (G) content is a common result in vertebrate mtDNA (e.g., AF531730, AF531715, D83636, AF531717, AF531718, Tamura and Nei 1993). AF531719, AF531720, AF531721, AF531735, AF531736, Of the 49 haplotypes found in this study, a BLAST search AF531737, AF531738, AF531740, AF531739, AF531741, conducted against the sequences in GenBank revealed that 40 D83611, D83637, AB007381) were imported from GenBank are new or unique haplotypes. Differences between the 40 to represent the 6 major dog clades described by Savolainen new haplotypes and the dog sequences deposited in Gen- and others (2002). These haplotypes represent 20 of the 75 Bank ranged from 1 to 15 nucleotides in 887 bp (0.1– haplotypes described by Savolainen and others (2002) for 1.7%). Twelve of the 49 haplotypes found in this study clade A and all haplotypes described for clades B, C, D, are shared among the 13 dog populations sampled, and E, and F. Haplotypes from wolves, golden jackal, and coyote the remaining 37 haplotypes are breed or dog population spe- were also included (accession numbers: AF008140, cific (Table 1). The total number of haplotypes found per AF008137, AF008142, AF008135, AF008138, AF008139, breed or dog population (shared and nonshared) varies be- AF008141, AF184048, AF008158). From the 3 Iberian wolf tween 1 and 13 (Table 1). The number of nonshared haplo- haplotypes described by Vila` and others 1997 (W1, W2, and types, that is, haplotypes that are only found in 1 of the 13 W3), we selected W1 as a representative of wolves from Ibe- dog groups of this study varies between 0 and 7 (Table 1). ria. Among W1, W2, and W3, there are only 5 variable posi- Within the sampled individuals, 11 haplotypes, 4 from Aidi, tions out of 261. In order to construct an NJ tree integrating 5 from Sloughi, and 2 from Tunisian dogs, were only found sequences from the studies mentioned above, the 887-bp in dogs from North Africa (Table 1). With the exception of fragments generated in this study were trimmed to 554 bp. the Portuguese Warren hound, Aidi and Sloughi were the The best-fitting model of sequence evolution was determined breeds showing the highest number of nonshared haplotypes by MODELTEST version 3.06 (Posada and Crandall 1998). (4 and 5, respectively, Table 1). Portuguese village dogs The reliability of the nodes was assessed with 1 000 bootstrap showed the highest value of haplotypic diversity (0.97 ± iterations (Felsenstein 1985). 0.04), followed by Aidi (Table 2). Among the remaining An intraspecific gene genealogy was inferred using the breeds, only the Portuguese Warren hound and Alentejo median-joining network algorithm (Bandelt and others 1999) Shepherd dog showed haplotype diversities higher than in NETWORK, version 4.1, available at http://www. 0.90. Haplotype diversity was not correlated to sample size fluxus-engineering.com. Gaps were treated as evolutionary (R2 5 0.0324, graph not shown). Among the endangered events, and data were analyzed with all characters weighing Portuguese native dogs, Portuguese Water dog had a relatively equally. The tolerance parameter e was set to 0. Haplotype high haplotypic diversity (0.82 ± 0.08). frequencies were converted into proportional areas in the Nucleotide diversity per site (p ± SD) was on average graphical output. high, ranging from 0.014 ± 0.007 (Estrela Mountain dog) to 0.002 ± 0.001 (Portuguese Pointer), with the exception Results of the Castro Laboreiro Watchdog that showed no diversity (Table 2). This statistic is not highly sensitive to sample size, Genetic Diversity and Differentiation as already noted by Vila` and others (1999). Castro Laboreiro The primers L15210/H16097 amplified a sequence compris- Watchdog showed a single and private or breed-specific hap- ing part of the cytochrome b gene (112 bp), the tRNA-Thr lotype (H25) in the 14 analyzed animals, of which 11 were and tRNA-Pro genes (135 bp), and the hypervariable portion working dogs and 3 were show dogs. Estrela Mountain at the 5# end of the control region (640 bp). Forty-nine dif- dog, Alentejo Shepherd dog, and Spanish mastiff showed ferent haplotypes were obtained from 164 individuals (acces- similar values of genetic diversity. It is noteworthy that high sion numbers: AY706476–524) (Figure 1). Forty-eight levels of nucleotide diversity were also found in the Northern phylogenetically informative sites were found (5% of the total African dogs (Table 2).

! Figure 1. Base substitutions and gaps found among 164 dog mitochondrial sequences. Forty-nine haplotypes are listed. Dashes (-) indicate gaps, and dots (.) indicate matches regarding the reference nucleotide sequence (Haplotype 10, the most frequent). Position 1 corresponds to the base 15211 on the cytochrome b gene in the mitochondrial genome sequence of Kim and others (1998). Nucleotide sequence data from this study have been assigned the GenBank accession numbers AY706476–524.

322 Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs

323 Journal of Heredity 2006:97(4)

Genetic differentiation among breeds and dog popula- 2 4.82 tions was statistically significant (v 5 1177.675, P ,

0.001). Pairwise FST ranged from nearly 0 (between Aidi PortVillage Dogs 9.79 ± and Portuguese Village dogs) to 0.801 (between Castro Laboreiro Watchdog and Portuguese Pointer) (Table 2). Pair- 0.004 0.011 ± 0.006

3.12 wise FST values between Portuguese village dogs and dogs from outside of Portugal (Spanish mastiff, Aidi, Sloughi, 0.016 5.92 ± and Tunisia dogs) were near 0. Castro Laboreiro Watchdog and Portuguese Sheepdog were the only breeds with all pairwise F values statistically significant. Castro Laboreiro

4.95 ST values (below the diagonal).

ST Watchdog, Portuguese Sheepdog, Portuguese Pointer, and 0.018 9.90 ± Portuguese Water dog, the least diverse breeds, showed the greatest genetic differentiation values from the Portu- guese village dogs (significant at the 5% level), which repre- 4.79 sent the ‘‘background’’ mtDNA variation of dogs in Portugal. 0.004 9.54 ± Within the livestock guarding dogs group (Castro Laboreiro Watchdog, Estrela Mountain dog, Alentejo Shepherd dog, Spanish mastiff, and Aidi), Castro Laboreiro Watchdog 3.53 was the most isolated, with no mtDNA variation and signif- 0.014 0.035 0.00 0.040 0.034 icant FST values for all breed comparisons (a 5 0.05) (all FST 6.87 ± values 0.396). Estrela Mountain dog and Alentejo Shep- herd dog showed little genetic differentiation from each 4.36 other. Spanish mastiff showed substantial differentiation from all Portuguese livestock guarding dogs, although to 0.305 8.71 ± 0.223 0.320 0.175 a much lesser extent from Estrela Mountain dog and Alentejo

4.90 Shepherd dog. FST between Spanish mastiff and Aidi (the only African livestock guarding dog in this study) was not significant (0.040). The insular Azores cattle dog shows the least 0.070 10.420 ± 0.103 0.038 0.013 differentiation from the Alentejo Shepherd dog (FST 5 0.027 and not significant). Portuguese Warren hound showed sig-

1.01 nificant FST values with the breeds Portuguese Sheepdog, Castro Laboreiro Watchdog, Portuguese Pointer, and Azores 0.103 0.109 1.53 ± 0.187 0.016 0.098 0.058 0.020 cattle dog. All FST values among Portuguese Warren hound subpopulations (varieties) were near 0 (data not shown).

1.93 FST values between each breed and the dogs that compose the background genetic population (Portuguese village dogs) 3.38 ± 0.218 0.191 0.223 0.197 0.125of 0.144 their region ranged from 0.066 to 0.073, and none were significant (data not shown). AMOVA showed subdivision

4.11 between breeds (UST 5 0.171, P , 0.0001), with around 80% of the variation found within populations (breeds). Var- 0.093 0.135 0.241 8.33 ± 0.118 iation among breeds accounted for practically 20% of the total genetic variability found. No variation could be attributed to geographic structure for the entire data set. 4.51 0.027 9.25 ± 0.138 0.173 0.215 0.199 0.278 0.410Phylogenetics 0.504

5.87 The best-fitting model of sequence evolution determined by MODELTEST (hierarchical likelihood ratio test statistic) was 0.014 0.053 0.102 12.26 ± 0.055 0.039 0.055 0.048 0.058 0.071the 0.081 HKY85 0.062 þ I þ G model (Hasegawa and others 1985) with gamma distribution correction for rate heterogeneity among sites. The shape parameter of the distribution (a) and propor- 0.00 tion of invariable sites (I) were 0.6306 and 0.8646, respectively. Only clades with bootstrap values equal or higher 0.801 0.317 0.359 0.401 0.415 0.00 ± 0.00 0.90 ± 0.050.000 ± 0.000 0.92 0.014 ± ± 0.04 0.007 0.010 ± 0.006 0.66 ± 0.09 0.009 ± 0.005 0.25 0.004 ± ± 0.18 0.002 0.002 ± 0.001 0.50 ± 0.13 0.012 ± 0.006 0.91 0.010 ± ± 0.04 0.006 0.008 ± 0.005 0.82 ± 0.08 0.011 ± 0.006 0.84 0.011 ± ± 0.10 0.006 0.007 ± 0.96 ± 0.06 0.89 ± 0.08 0.83 ± 0.13 0.97 ± 0.04 0.473 0.513 0.799 0.269 0.227 0.296 0.403 0.518 0.163 0.228 0.230 0.237 0.472 0.473 0.148 0.152 0.00 ± 0.550 0.136 0.460 0.674 0.396 CLWD EMD ASD ACD PSD PP PWH PWD SM Aidi SL Tun than 70% are considered here (see Hillis and Bull 1993). Haplotype diversity, nucleotide diversity, and average pairwise differences between haplotypes within populations (underlined values) and F Figure 2 shows an NJ unrooted phylogram of all haplotypes found in the samples of this study (887 bp). Mitochon-

±SD) drial lineages are not clustered by geographic origin or ±SD) H p PP Table 2. Haplotype diversity ( Nucleotide diversity ( CLWD Aidi ASD PSD PWH Tun EMD SM ACD SL PWD PortVillageDogs Significant values at the 5% level are in bold. Abbreviations of breed names as in Table 1 traditional breed classification: the same haplotypes occur

324 Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs

H45 H31 H49 H30

H17 H11 H12 H34 H9 H33 H26 92 H22

H16 H2 H29 H38 99 H27 H39 82 H23 H5 84 H32 H6 H40 H37 83 H3

H21 H18 H15 H36 H41 H24 H28 H46 H4 H19 H13 H1 H42 H8 H7 H10 H44 H25 H48H43 H35 H20 0.0005 substitutions/site H14 H47

Figure 2. Unrooted NJ phylogram of dog haplotypes based on 887 bp, comprising partial sequences of cytochrome b, tRNA- Thr, tRNA-Pro, and control region. Letters A–D indicate clades consistently supported with different tree-building methods (data not shown). Support values are indicated at nodes when found in at least 70% of 1000 bootstrap replicates. in different populations and are scattered throughout differ- (AY706481) obtained from Morocco. For the other clades, ent clades. Forty-nine percent of the haplotypes found in this 12 new haplotypes were found: in clade A, 5 (Portugal and study were in clade A and with all breeds represented, al- Morocco, H19, H25, H41, H42, H44); clade B, 3 (Portugal though with weak bootstrap support. Clades B, C, and D and Morocco, H30, H34, H45); and clade C, 4 (Portugal and are supported by a high bootstrap value (83%) and include Spain, H16, H27, H29, H32) (AY706494, AY706500, all breeds except Castro Laboreiro Watchdog (H25). Clade AY706516, AY706517, AY706519, AY706505, AY706509, D, previously described as being regionally restricted to AY706520, AY706491, AY706502, AY706504, AY706507, Turkey, Spain, and Scandinavia (Savolainen and others respectively). The following haplotypes were breed or 2002), includes Estrela Mountain dog and Alentejo Shepherd population-specific within this study: Castro Laboreiro dog haplotypes from Portugal (H38 and H40) and Aidi hap- Watchdog, 1 (H25); Estrela Mountain dog, 2 (H41, H42); lotypes from Morocco (H2 and H6) (AY706513, AY706515, Portuguese Warren hound, 4 (H29, H30, H32, H34); Spanish AY706477, and AY706481 respectively) (Figures 2 and 3). mastiff, 1 (H27), Aidi, 1 (H6); Sloughi, 2 (H44, H45) and Por- Few nodes showed significant bootstrap values, but most tuguese village dogs, 2 (H16, H19) (Table 1). The W1 Iberian major partitions are well supported. When 54 domestic wolf haplotype grouped with dog haplotypes from clade B, dog haplotypes from Savolainen and others (2002) and wild along with haplotypes from all breeds except Castro canid sequences from Vila` and others (1997) (fragments of Laboreiro Watchdog, Portuguese Sheepdog, Aidi, and Por- 554 bp) were included, the structure of the NJ tree is very tuguese Pointer. The C. aureus and C. latrans sequences were similar to that obtained by Savolainen and others (2002) (data both highly distinct, as expected. not shown). We recovered all clades except E and F. Regard- The haplotype network (Figure 3) shows the same rela- ing the 554-bp sub–data set of the sequences generated in this tionships as the NJ tree (Figure 2), but more detail is evident. study, we identified one new haplotype for clade D Eight missing haplotypes (median vectors, mv) were detected.

325 Journal of Heredity 2006:97(4)

Figure 3. Median-joining network (e 5 0) depicting phylogenetic relationships among, and breed assignment of, all dog breed mtDNA haplotypes from this study. Circled areas are proportional to the corresponding haplotype frequency. If individuals of different breeds shared one haplotype, a pie diagram illustrates the respective proportions. Reticulations are observed. Median vectors (mv) produced by the network software, representing missing or not sampled haplotypes, are illustrated by black dots.

In clade A, all breeds are represented, but 4 median vectors Discussion are also included. The most frequent haplotypes, H7 and H10 Genetic Diversity and Differentiation (AY706482 and AY706485), respectively, are present in 16 (7 dog populations) and in 19 (6 dog populations) animals. Because most haplotype diversities were high (Table 2), we These frequent haplotypes are connected to other haplo- would expect the v2 test implemented in Chiperm to be pow- types, from which they mostly differ by singletons. Haplo- erful in detecting genetic differentiation between breeds or type H25 (AY706500) in clade A is only one mutational populations (Hudson and others 1992). Genetic differentia- step (transition in position 422) different from haplotype tion was especially apparent due to the number of private H28 (AY706503). Haplotype H28 is shared by the Portu- haplotypes found in several breeds. The lack of differentia- guese Pointer, Alentejo Shepherd dog, and Portuguese War- tion between Portuguese village dogs and dogs from outside ren hound breeds. Clade A is connected to Clade C by 11 Portugal (Spanish mastiff, Aidi, Sloughi, and Tunisia dogs) is mutational steps, including 2 median vectors. Clade C is con- probably a consequence of the high diversity found in all nected to Clade B by the same number of steps. In the net- these breeds and/or populations. work, Clade D comprises 4 divergent lineages and is Portuguese Warren hound subtypes (varieties) are not dif- separated from the rest of the haplotypes by 8 steps. This ferentiated using mtDNA. Portuguese Water dog was listed in clade comprises only 3 breeds in this study, whereas clades the Guinness Book of World Records in 1970 as the world’s B and C represent 9 and 8 dog populations, respectively. rarest dog breed (Molinari 1989) and has come close to ex- Haplotypes H7, H10, H21, and H5 were shared between Por- tinction twice in the last century, with a total of only 50 ani- tuguese and African breeds. mals in 1974 (Molinari 1993). However, the genetic diversity

326 Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs of Portuguese Water dog is surprisingly high. In Portuguese to have been more restricted. At the origin of the Azores Kennel Club books, registrations for this breed have been cattle dog, the Portuguese native breeds Castro Laboreiro less than 50 animals per year for 40 years and have never Watchdog, Estrela Mountain dog, and Alentejo Shepherd exceeded 450 animals per year. Although this breed is very dog are considered founders (Amaral and Veiga 2004). popular in the United States, only one individual could be Our results only support a major contribution by the Alentejo sampled from there (a dog imported from the United States Shepherd dog because the FST value between the Azores and living in Portugal), and other Portuguese Water dogs liv- cattle dog and the Alentejo Shepherd dog is the lowest value ing in Portugal share its haplotype (H11). Further efforts and is not significant. need to be undertaken to test more individuals from lineages It is interesting to note that livestock guarding and herd- exported to the United States from Portugal in the 1960s ing dog breeds, namely Estrela Mountain dog, Alentejo Shep- (Molinari 1993). Concerning Castro Laboreiro Watchdog, herd dog, and Azores cattle dog, do not have a statistically even though it is a livestock guarding dog like Estrela Moun- significant divergent mtDNA composition from that of tain dog, Alentejo Shepherd dog, and Spanish mastiff, it was the ‘‘background’’ gene pool in the region where they are never involved in the large transhumance movements in from, probably due to the high haplotypic diversity revealed which all other livestock guarding dog breeds participated by the Portuguese village dog population. Finally AMOVA (Geraldes 1996) and has been isolated for long periods. It results suggest that, although breeds share haplotypes, there was, instead, involved in small, geographically restricted is breed differentiation. Further sampling in Spain and North migrations within a remote area, and contact with other Africa may shed more light on the geographic structure of dog populations is likely to have been scant. This is a possible Iberian and North African dog breeds. explanation for its mtDNA uniqueness. Although working The high levels of diversity in African dogs could be animals lacking the breed standard phenotype can be found explained by their antiquity and/or reported introgression. in the area, these animals were not sampled for this study. Both Aidi and Sloughi are considered rare (Miguil 1986; Ouar However, they might be a possible addition to future analy- 1994). At the beginning of the 20th century, most of the dog ses. Expected heterozygosity values inferred using microsat- population in the Atlas mountainous area comprised Aidi ellites loci indicate that Castro Laboreiro Watchdog is one of dogs. In contrast, by 1994, only 49% of the dog population the least diverse breeds in Portugal (Amaro 2001; AE Pires, corresponded to Aidi breed individuals, and in parallel with F Simo˜es, F Petrucci-Fonseca, and MW Bruford, in prepara- a reduction in numbers, there was documented introgression tion). Within the dogs sampled for this study, position 422 with hunting dogs (Ouar 1994). This breed may also be less shows a transition (A to G) that separates Castro Laboreiro intensively managed than many European breeds. The breed Watchdog from all other dogs. Therefore, this nucleotide can standard was described in 1969, but the majority of animals provisionally be considered a breed-specific marker in the are free ranging for guarding purposes (Ouar 1994), making study area. As can be seen in the network (Figure 3), this hap- possible introgression with feral dogs and/or other dog lotype differs by one base from a more common haplotype breeds. The date of origin of the Sloughi remains speculative, present in several dog breeds (Portuguese Pointer, Portu- but it is considered an ancient breed. Representatives of Af- guese Warren hound, and Estrela Mountain dog). Probably rican Sighthound-like dogs date back to more than 7000 years Castro Laboreiro Watchdog as it is today, an mtDNA mono- (Giudicelli 1975). At present, it is mainly used to hunt desert morphic dog breed, is the result of a severe bottleneck from hares and gazelles and indirectly to protect goat and sheep a more diverse ancestral population of Castro Laboreiro herds by hunting their predator, the golden jackal (Miguil Watchdogs. Considering all haplotypes from Savolainen 1986). A number of factors have contributed to its present and others (2002) and Vila` and others (1997), the guanine low population size, including a decrease of hunting activities in position 422 is found in one dog from East Asia (isolate: using dogs, the popularity of firearms, and a reduction in its A56, accession number: AF531707). However, the Castro geographic distribution (Miguil 1986). Finally, the high ge- Laboreiro Watchdog haplotype remains unique when com- netic diversity observed in Tunisian dogs is expected because pared with this East Asian dog due to a difference in another the dogs sampled belong to the local village dog population nucleotide position (position 430). Although less likely, from no specific breed. Tunisian dogs show considerably less this haplotype may represent a lineage tracing back to the differentiation from the other African dogs and from the livestock guarding dogs dispersal from Southwest Asia (as- Spanish mastiff than from the Portuguese native dog breeds. sociated with human migrations), where livestock was first African domestic dogs were found to have distinct haplo- domesticated (Mannion 1999). types and haplotype frequencies from Portuguese dogs, The low levels of genetic differentiation between Estrela and although some haplotypes are shared, no south–north Mountain dog and Alentejo Shepherd dog are expected based cline in frequency could be detected in our data as found on historical information: these breeds have a probable com- in cattle, for which admixture was detected by Cymbron mon origin, and until recently both have been in close contact and others (1999). due to the transhumance practice (Alpoim 1999). In contrast, genetic differentiation between Spanish mastiff and both Phylogenetic Analysis Estrela Mountain dog and Alentejo Shepherd dog is more pronounced as genetic exchange that might have occurred The lack of an obvious underlying geographic or breed- during the long period of extensive transhumance is likely related structure in the NJ tree could indicate that most of

327 Journal of Heredity 2006:97(4) the breeds are derived from the same ancestral stock and re- of Vila` and others (2005) who analyzed variation in genes tain many of the ancestral maternal haplotypes and/or that from the major histocompatibility complex (MHC) poten- extensive introgression has occurred in the process of breed tially under balancing selection to infer past relationships be- formation and development. Similar results were obtained by tween domestic mammals and their wild ancestors. The Tanabe and others (1991) based on blood proteins; Okumura authors found high levels of genetic diversity suggesting that and others (1996), Tsuda and others (1997), Vila` and others domestic mammals might have acquired much of their cur- (1997), and Savolainen and others (2002) based on mtDNA rent genetic diversity by backcrossing several times with their control region; and Jordana and others (1999) using morpho- wild ancestors. Typing nuclear genes from the MHC in each logical and behavioral characteristics. Another explanation of the breeds found to contain unique mtDNA haplotypes in may be that most breeds have originated too recently, and our study would probably reveal additional MHC alleles. mutations that account for breed differentiation have yet to occur and become fixed. Other researchers have found dog breeds to be highly differentiated. For example, Parker Implications for Conservation and others (2004) sampling purebred dogs from closed gene Dog breeds should be viewed as dynamic populations, in pools found that breeds could be genetically differentiated which historic phenomena such as admixture, introgression, using biparentally inherited microsatellites markers. How- genetic isolation, and drift have occurred (Neff and others ever, by sampling only 5 unrelated pedigreed animals from 2004). Portuguese dog breeds have experienced major demo- each breed, the authors did not carry out an extensive and graphic fluctuations over the past 20 generations, with a con- representative sampling across each breed, thereby maximiz- sistent decline through disuse of breeds in a working context, ing the probability of detecting a signal of breed structure. followed by a rapid expansion in many cases due to the fash- Most of the haplotypes in this study clustered within ion of showing dogs and for the pet trade. Concerning its the 3 major phylogenetic clades A, B, and C described in mtDNA, Iberian and North African breeds show, in general, Savolainen and others (2002), implying that Iberian dogs considerable diversity, including the possession of some are unlikely to be the result of an additional domestication newly described sequences. In this study that focused on event. All clades found by Savolainen and others (2002) were marginal working dog breeds, several haplotypes emerge extensively represented in the phylogenetic analysis, although as novel, and conservation planning for these breeds should the proportion in clade A (26.6%) was reduced relative to therefore take into account the unique genetic characters their study. Therefore, the 8 breed-specific haplotypes found highlighted here. Following Weitzman’s concept for live- in Iberian and North African dogs for clades B, C, and D stock that involves the use of a ‘‘diversity function’’ (Bruford represent additional diversity in these clades. This unique and others 2003), Castro Laboreiro Watchdog and Portu- diversity is potentially important information to the conser- guese Sheepdog would become priorities for conservation vation of domestic dog genetic variation in this region. on the basis of mtDNA alone. The considerable mtDNA di- The application of single-genetic locus surveys is not versity in the dogs of Iberia and North Africa prevents, with without problems (Avise 2004), and this work should be aug- one exception, its use as a marker for breed designation, mented by neutral nuclear DNA markers and genes associ- and these dog breeds should be studied with several other ated with the phenotypes under selection. Currently, studies kinds of nuclear markers available to accurately assess breed based on nuclear DNA markers such as amplified fragment distinctiveness. length polymorphisms and autosomal microsatellites are on- going (AE Pires, F Simo˜es, F Petrucci-Fonseca, and MW Bruford, in preparation). MtDNA does not account for Acknowledgments male-mediated gene flow, a dominant force in domestic an- The research was supported by Fundacxa˜o para a Cieˆncia e a Tecnologia (grant imal evolution (Petit and others 2002). Male dog microsatel- PRAXIS XXI/BD/21677/99) and AGRO (project 31106). We thank Carla lites loci from the nonrecombining portion of the canine Y Cruz, Silvia Ribeiro, the dog owners, and breed clubs who contributed to this chromosome are already available (Olivier and others 1999; study; Margarida Gomes for helping collecting data from the Portuguese Bannash and others 2005), and their analysis among breeds Kennel Club; Carlos Fernandes for advice in the laboratory and data analyses; revealed a high breed and geographic structure (Bannash and Isabel Amorim, Carlos Fernandes, 2 anonymous reviewers, and the corresponding editor for helpful discussions and comments on the manuscript. others 2005). Similarly, it is likely that the study of the Y chro- We also thank the Portuguese Kennel Club for facilitating access to the da- mosome can bring additional detail to the phylogenetic anal- tabase on dog registries. ysis of the domestic dog breeds analyzed here. Other possible avenues of research involve the analysis of functional genes. For example, Fondon and Garner (2004) studying the associ- References ation of DNA mutations, in particular intragenic tandem Alpoim JA. 1999. O Rafeiro do Alentejo. Monografia da Racxa [Alentejo repeats in developmental genes, with phenotypic variability, Shepherd Dog. Breed monography]. Monforte, Portugal: Caˆmara Municipal allowed identification of a possible molecular mechanism de Monforte. responsible for morphological and functional variation. Neff Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local and others (2004) by using a pharmacogenetic mutation as alignment search tool. J Mol Biol 215:403–10. a molecular marker were able to unravel the emergence of Amaral AJ, Veiga V. 2004. O Ca˜o de Fila de S. Miguel [The Azores cattle dog]. formally recognized breeds. Another example is the work Vila Franca do Campo, Portugal: Caˆmara Municipal de Vila Franca do Campo.

328 Pires et al. Mitochondrial DNA of Portuguese Domestic Dogs

Amaro A. 2001. Contribuicxaõ para a Caracterizacxaõ da Estrutura Gene´tica de ICN, 1997. Conservacxaõ do lobo em Portugal [Wolf conservation in Portu- Racxas Caninas Autoćtones Portuguesas. Estudo do Grupo de Ligacxaõda gal]. Lisboa, Portugal: Instituto da Conservacxaõ da Natureza. 231 p. Transferrina em Racxas Caninas Tipo Mastim [Contribution to the genetic Jordana J, Manteca X, Ribo O. 1999. Comparative analysis of morphological characterization of the Portuguese autochthonous dog breeds. Study of and behavioral characters in the domestic dog and their importance in the transferrin ligation group in mastiff type breeds] [MSc dissertation]. Porto, reconstruction of phylogenetic relationships in canids. Genet Mol Biol Portugal: Universidade do Porto. 22:49–57. Avise JC. 2004. Molecular markers, natural history and evolution. Sunder- Kim KS, Seong EL, Jeong HW, Ha JH. 1998. The complete nucleotide se- land, MA: Sinauer Associates, Inc. quence of the domestic dog (Canis familiaris) mitochondrial genome. Mol Bandelt H-J, Forster P, Ro¨hl A. 1999. Median-joining networks for inferring Phylogenet Evol 10:210–20. intraspecific phylogenies. Mol Biol Evol 16:37–48. Lorenz K. 1975. Foreword. In: Fox MW, editor. The wild canids: their Bannash DL, Bannash MJ, Ryun JR, Famula TR, Peterson NC. 2005. systematics, behavioural ecology and evolution. New York: Van Nostrand Y chromosome haplotype analysis in purebred dogs. Mamm Genome 16: Reinhold. 273–80. Mannion AM. 1999. Domestication and the origins of agriculture: an ap- Braga PD. 2000. Histo´ria dos Ca˜es em Portugal. Das Origens a 1800 [Dogs’ praisal. Prog Phys Geogr 23:37–56. history in Portugal from origin to 1800]. Lisboa, Portugal: Hugin Editores, Martıń PG, Angioni G, Raverdy Ch, Ojanen U, Este´banez NL, Martıńez BG, Lda. Chamorro GV, Lenberg JG, Ilera FA, Alonso AC, Albaladejo PF, Bernal Brito RS, Fabiaõ C, Macıás S, Mattoso J, Torres C. 1992. Histo´ria de Portu- AM, Toledo AA, Dıéz LM, Canada AM, Garcıá MM, Pascual MR, Cardoso gal. Antes de Portugal [History of Portugal. Before Portugal]. Volume 1. JJ. 1994. Por los Caminos de la Trashumancia [On the Tanshumance paths]. Lisboa, Portugal: Cı´rculo de Leitores, Lda and Authors. León, Spain: Junta de Castilla y León. Bruford MW, Bradley DG, Luikart G. 2003. DNA markers reveal the com- McDonald DW, Carr GM. 1995. Variation in dog society: between resource plexity of livestock domestication. Nat Rev Genet 4:900–10. dispersion and social flux. In: James Serpell, editor. The domestic dog: its Cardoso JL. 2002. Pre´-Histo´ria de Portugal [Pre-history of Portugal]. Lisboa, evolution, behaviour and interactions with people. Cambridge: Cambridge Portugal: Editorial Verbo. University Press. p 199–216. Clutton-Brock J. 1984. Dog. In: Mason IL, editor. Evolution of domesticated Miguil A. 1986. Contribution a l’e´tude du Sloughi au Maroc [PhD disserta- animals. London, UK: Longman. tion]. Rabat, Morocco: Institut Agronomique et Veterinaire Hassan II. Coppinger R, Coppinger L. 2002. Dogs: a new understanding of canine or- Molinari C. 1989. El Cao de Aguas Portugues. Um perro pescador [The igin, behavior, and evolution. Chicago, IL: University of Chicago Press (Trd). Portuguese Water dog. A fisher dog]. La Revista del Perro No. 29. Madrid, Spain: Ediciones Guau, SA. Cymbron T, Loftus RT, Malheiro MI, Bradley DG. 1999. Mitochondrial sequence variation suggests an African influence in Portuguese cattle. Proc Molinari C. 1993. The Portuguese Water dog. 1st ed. Mafra, Portugal: Author. R Soc Lond B Biol Sci 266:597–603. Neff MW, Robertson KR, Wong AK, Safra N, Broman KW, Slatkin M, Excoffier L, Smouse P, Quattro JM. 1992. Analysis of molecular variance Mealey KL, Pedersen NC. 2004. Breed distribution and history of inferred from metric distances among DNA haplotypes: application to hu- canine mdr1-1D, a pharmacogenetic mutation that marks the emergence man mitochondrial DNA restriction data. Genetics 131:479–91. of breeds from the collie lineage. Proc Natl Acad Sci USA 101: 11725–30. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–91. Okumura N, Ishiguro N, Nakano M, Matsui A, Sahara M. 1996. Intra- and interbreed genetic variations of mitochondrial DNA major non-coding Fondon JW III, Garner HR. 2004. Molecular origins of rapid and continuous regions in Japanese native dog breeds (Canis familiaris). Anim Genet morphological evolution. Proc Natl Acad Sci USA 101:18058–63. 27:397–405. Frankham R, Ballou JD, Briscoe DA. 2002. Introduction to conservation Olivier M, Breen M, Binns MM, Lust G. 1999. Localization and character- genetics. Cambridge, UK: Cambridge University Press. ization of nucleotide sequences from the canine Y chromosome. Chromo- Geraldes AD. 1996. Brandas e inverneiras: particularidades do sistema some Res 7:223–33. agro-pastoril crastejo [Particularities of the Castro Laboreiro’s agricultural Ostrander EA, Comstock KE. 2004. The domestic dog genome. Curr Biol and pastoral system]. Lisbon, Portugal: Instituto da Conservacxaõda 14:R98–9. Natureza/Parque Nacional da Peneda-Gereˆs. Ouar F. 1994. Contribution a l’e´tude de la situation du chien de l’Atlas: Giudicelli B. 1975. Situation Actuelle du Sloughi en Algerie [PhD disserta- re´partition geógraphique, ećologie et caracte´ristiques morphome´triques tion]. Lyon, France: Ecole Nationale Ve´te´rinaire. [PhD dissertation]. Rabat, Morocco: Institut Agronomique et Veterinaire Gomes MS. 2003. Racxas Caninas Autoćtones Portuguesas. Contributo para o Hassan II. seu Estudo Gene´tico e Demogra´fico [Portuguese autochthonous dog breeds. Parker HG, Kim LV, Sutter NB, Carlson S, Lorentzen TD, Malek TB, A contribution to their genetic and demographic study] [Licenciatura disser- Johnson GS, DeFrance HB, Ostrander EA, Kruglyak L. 2004. Genetic tation]. Santare´m, Santare´m: Escola Superior Agra´ria de Santare´m. structure of the purebred domestic dog. Science 304:1161–4. Graur D, Li W-H. 2000. Fundamentals of molecular evolution. 2nd ed. Petit E, Ballou F, Excoffier L. 2002. Mammalian population genetics: why Sunderland, MA: Sinauer Associates. not Y? Trends Ecol Evol 17:28–33. Hasegawa M, Kishino H, Yano T. 1985. Dating of the human-ape splitting by Petrucci-Fonseca F, Pires AE, Ribeiro S, Almendra L, Clemente A, Collacxo a molecular clock of mitochondrial DNA. J Mol Evol 22:160–74. MT, Matos J, Simo˜es F. 2000. Ca˜es de gado e a conservacxaõ do lobo em Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for Portugal [Livestock guarding dogs and wolf conservation in Portugal]. assessing confidence in phylogenetic analysis. Syst Biol 42:182–92. Galemys 12:135–48. Hoelzel AR, Hancock JM, Dover GA. 1991. Evolution of the cetacean Posada D. 2000. Testing geographic association—Chiperm. Version 1.2. mitochondrial D-Loop region. Mol Biol Evol 8:475–93. Provo, UT: Department of Biology, Brigham Young University. Hudson RR, Boos DD, Kaplan N. 1992. A statistical test for detecting geo- Posada D, Crandall KA. 1998. MODELTEST: testing the model of DNA graphic subdivision. Mol Biol Evol 9:138–51. substitution. Bioinformatics 14:817–8.

329 Journal of Heredity 2006:97(4)

Ribeiro A, Saraiva JH. 2004. Histo´ria de Portugal. A Formacxaõdo Tsuda K, Kikkawa Y, Yonekawa H, Tanabe Y. 1997. Extensive interbreeding Territo´rio—da Lusitaˆnia ao Alargamento do Paı´s [History of Portugal. occurred among multiple matriarchal ancestors during the domestication of Territory formation—from Lusitania to country expansion]. Matosinhos, dogs: evidence from inter- and intraspecies polymorphisms in the D-loop Portugal: Quidnovi. region of mitochondrial DNA between dogs and wolves. Genes Genet Syst Roff DA, Bentzen P. 1989. The statistical analysis of mitochondrial DNA 72:229–38. 2 polymorphisms: v and the problem of small samples. Mol Biol Evol 6: Vasconcelos RC. 1995. Racxas de Ca˜es Portugueses [Portuguese dog breeds]. 539–45. 2nd ed. Lisboa, Portugal: Editorial Presencxa. Roger L, Boxshall G, Clark P. 1998. A dictionary of ecology, evolution and Veiga V. 2001. O Podengo Portugueˆs [Portuguese Warren hound breed]. systematics. 2nd ed. Cambridge, UK: Cambridge University Press. Lisboa, Portugal: Author. Sambrook J, Russel DW. 2001. Molecular cloning—a laboratory manual. 3rd Vila` C, Amorim IR, Leonard JA, Posada D, Castroviejo J, Petrucci-Fonseca ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. F, Crandall KA, Ellegren H, Wayne RK, 1999. Mitochondrial DNA phylo- Savolainen P, Zhang YP, Luo J, Lundeberg J, Leitner T. 2002. Genetic geography and population history of the grey wolf Canis lupus. Mol Ecol evidence for an East Asian origin of domestic dogs. Science 298:1610–3. 8:2089–103. Schneider S, Roessli D, Excoffier L. 2000. Arlequin version 2.000: a software Vila` C, Savolainen P, Maldonado JE, Amorim IR, Rice JE, Honeycutt RL, for population genetics data analysis. Geneva, Switzerland: Genetics and Crandall KA, Lundeberg J, Wayne RK. 1997. Multiple and ancient origins of Biometry Laboratory, University of Geneva. the domestic dog. Science 276:1687–9. Shields GF, Kocher TD. 1991. Phylogenetic relationships of North Vila` C, Seddon J, Ellegren H. 2005. Genes of domestic mammals augmented American ursids based on analysis of mitochondrial DNA. Evolution 45: by backcrossing with wild ancestors. Trends Genet 21:214–8. 218–21. Walsh PS, Metzger DA, Higuchi R. 1991. Chelex 100 as a medium for single Silva RF. 2000. Transumaˆncia no Portugal central: diversidade e organizacxaõ extraction of DNA for PCR-based typing from forensic material. Biotech- do territo´rio [Transhumance in central Portugal: diversity and land organi- niques 10:506–13. zation]. In: Associacxaõ da defesa do patrimońio Arouquense, editor. Montemuro—au´ltima rota da tansumaˆncia. Arouca: Associacxaõ da defesa Wayne RK. 1986. Cranial morphology of domestic and wild canids: the in- do patrimońio Arouquense, Escola Superior Agra´ria de Viseu. p 55–69. fluence of development on morphological change. Evolution 40:243–61. Southern SO, Southern PJ, Dizon AE. 1988. Molecular characterization of Yang Z. 1994. Maximum likelihood phylogenetic estimation from DNA a cloned dolphin mitochondrial genome. J Mol Evol 28:32–42. sequences with variable rates over sites: approximate methods. J Mol Evol 39:306–14. Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates. Zimen E. 1981. The wolf: his place in the natural world. London: Souvenir Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitu- Press. tions in the control region of mitochondrial DNA in humans and chimpan- zees. Mol Biol Evol 10:512–26. Received August 26, 2005 Tanabe Y, Oˆ ta K, Ito S, Hashimoto Y, Sung YY, Ryu JK, Faruque MO. 1991. Accepted May 4, 2006 Biochemical-genetic relationships among Asian and European dogs and the ancestry of the Japanese native dog. J Anim Breed Genet 108:455–78. Corresponding Editor: Robert Wayne

330