Journal of Heredity Advance Access originally published online on July 3, 2006
Journal of Heredity 2006 97(4):318-330; doi:10.1093/jhered/esl006
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Mitochondrial DNA Sequence Variation in Portuguese Native Dog Breeds: Diversity and Phylogenetic Affinities
From Centro de Biologia Ambiental, Faculdade de Ciências da Universidade de Lisboa, Rua Ernesto Vasconcelos, Edifício C2-3° Piso, 1749-016 Campo Grande, Portugal (Pires and Petrucci-Fonseca); Instituto Nacional de Engenharia, Tecnologia e Inovação, Estrada do Paço ao Lumiar, 22, Edifício E, Molecular Biology Group, 1649-038 Lisboa, Portugal (Pires and Matos); Laboratoire d'Analyses Génétiques Vétérinaires, Institut Agronomique et Vétérinaire 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: pireseg{at}cf.ac.uk.
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 monomorphic for mtDNA and possessed a new haplotype, which may be provisionally considered a breed-specific marker. Phylogenetic 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 familiaris, is the gray wolf Canis lupus. Gray wolves and dogs differ by only 4.6% (12 substitutions in a 261-bp fragment of the D-loop) in their mitochondrial DNA (mtDNA) (Vilà and others 1997), and this comparison is the lowest value between any pair of canid species. Other lines of evidence also place the wolf as the dog ancestor, such as morphology (Wayne 1986), behavior (Zimen 1981), and vocalization (Lorenz 1975; Zimen 1981).
Currently, the domestic dog is the most abundant canid, with a global population of around 400 million animals, and has the widest geographic distribution of any domestic species (Coppinger R and Coppinger L 2002). Among mammals, domestic dogs are remarkable by the range of natural variation they exhibit in terms of morphological and behavioral traits, even if the maintenance of dog breeds is guided by established standards, such that within each breed animals often show an uniform and distinctive morphology and behavior (Ostrander and Comstock 2004). There are more than 400 dog breeds worldwide, and their economic importance is substantial (Clutton-Brock 1984).
Although dogs were present in what is now Portugal by the Mesolithic (Cardoso 2002), the first breeds (probably used for hunting) are known from mosaic depictions dating from the Roman occupation (Braga 2000), and it is probable that the Phoenicians had previously brought hound-type dogs into the country (Veiga 2001). Currently, there are 10 working native dog breeds, recognized by the Portuguese Kennel Club, that as a group demonstrate remarkable phenotypic diversity. Seven of these dog breeds are also recognized by the Fédération Cynologique Internationale. These breeds are thought to have a relatively recent origin although their foundation dates are unknown. Breed standards date mostly from the first half of the 20th century, and intensive selection for standards started only 20–30 years ago.
Recent demographic changes are predicted to have impacted the genetic diversity of Portuguese breeds. For example, in the past, transhumance (twice-yearly migration of livestock, shepherds, and their various livestock guarding dog breeds [Martín and others 1994]), which has not occurred in Portugal since the 1990s (Silva 2000) and in Spain since the 1950s (Coppinger R and Coppinger L 2002), is likely to have facilitated gene flow among livestock guarding dog populations and breeds when they were brought into contact during these seasonal migrations. Further, some Portuguese dog breeds have gone through periods of critically low population size (Vasconcelos 1995; Gomes 2003) due to rural emigration, the abandonment of agricultural and hunting practices, and major reductions of wolf populations in some regions ICN (1997). With fewer wolves in the wild, shepherds began using dogs with no specific skills for livestock guarding, and the subsequent decrease in the use of livestock guarding dog caused a reduction in their number. Finally, the popularity of dog shows created subsets of demographically isolated dogs within some breeds (i.e., show vs. working dogs). Portuguese livestock guarding dogs are a good example of this, where both subsets have become distinct, not only morphologically (C. Cruz, personal communication) but also genetically (Petrucci-Fonseca and others 2000). Most of the demographic data collected from the Portuguese Kennel Club refers only to show dogs, and therefore, the number of registrations per year does not necessarily reflect the total population at any given time. Almost all breeds show a common pattern, concerning the evolution of their numbers, of an increase in the number of registries in the Portuguese Kennel Club by the end of the 1970s (Gomes 2003).
Spain and the Maghreb region (Morocco, Tunisia, and Algeria) are geographically proximate to Portugal and are all within the same zoogeographic region: the Mediterranean subregion of the Palaeartic (Roger and others 1998). Historically, the Iberian Peninsula has had a close connection with North Africa mainly due to the Moorish occupation, which lasted for 8 centuries (Brito and others 1992; Ribeiro and Saraiva 2004). The Moors introduced crops and livestock to Iberia (Cymbron and others 1999), and it is possible that livestock guarding dogs were traded as well. In order to assess genetic variation between domestic dog populations from North Africa and Iberia, the 2 internationally recognized North African dog breeds, the Aidi and Sloughi, and mongrel dogs from Tunisia were included in our study.
MtDNA is a powerful tool for estimating levels of genetic diversity, phylogenetic structure, and recent demographic history in domestic animals, but its use beyond these applications is more limited (Bruford and others 2003), especially because it only provides information about the female lineage (Avise 2004). This limitation is a drawback when studying domestic animals because male-mediated gene flow is usually more pronounced among them. For example, the application of mtDNA markers in domestic dogs (e.g., Okumura and others 1996; Tsuda and others 1997; Vilà and others 1997; Savolainen and others 2002) has showed little correspondence between mitochondrial lineages, geographic structure, or traditional breed classification, and many breeds contain haplotypes shared by other breeds scattered over different phylogenetic clades.
The most recent phylogenetic analysis of mtDNA in domestic dogs, including samples from Europe, Asia, Africa, and Arctic America, assigned mtDNA sequences to 6 clades: A, B, C, D, E, and F (Savolainen and others 2002). At least 5 wolf matrilines are considered to be present at the origin of the domestic dog population because all clades but F are intermingled with wolf sequences. Clades A, B, and C contain 95.9% of the sampled dog haplotypes and are represented in all geographic regions (clade A) or in all except America (clades B and C). Geographically more restricted are the clades D, E, and F, which, respectively, are found in Turkey, Spain, and Scandinavia; Japan and Korea; and Japan and Siberia.
This is the first comprehensive study of mtDNA variation of Portuguese dogs and some other geographically adjacent populations—the Spanish mastiff, Aidi, Sloughi, and Tunisian mongrel. Table 1 summarizes the function, country of origin, and conservation status of these breeds. The major aim of this investigation was to determine the mtDNA genetic variability of Portuguese dogs and to evaluate the geographic context of genetic variation in these and neighboring dog populations.
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Methods |
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 Top Methods ResultsDiscussionReferences |
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Sampling and DNA Extraction
Blood, hair, and tissue samples from 164 animals belonging to 13 different breeds or populations of dogs were collected at dog shows, from breeding kennels, and from distinct locations in their historical breed regions (Table 1). Animals were selected based on morphological standards and on information about their ancestry in order to exclude related animals back to the third generation whenever possible, although complete information on ancestry was not always available. In the Portuguese Warren hound, 3 body size types occur: small, medium, and large. Hence, individuals from this breed were sampled more extensively. Since the 1960s, one Portuguese Water dog breeder has exported dogs to outside of Portugal, mainly to the United States (Molinari 1993), and one animal from the United States is included, possibly representing an extinct lineage in Portugal. Samples were also collected from dogs kept in municipal kennels or animal shelters, whose phenotypes could not be assigned to any recognizable breed (hereafter Portuguese village dogs) as defined by McDonald and Carr (1995). We selected those individuals from diverse regions such as the Azores, where the Azores cattle dog originated, the Estrela Mountain region, where the Estrela Mountain dog originated, and Alentejo, where the Alentejo Shepherd dog originated. In Tunisia, there are no formally established dog breeds, and consequently, samples were collected randomly within the local population. To minimize the risk of both the effects of genetic relatedness among individuals and introgression from other breeds and/or village dogs, we sampled for each breed, working animals with unknown ancestry only when from remote regions within the historical range of each breed.
The number of registered females for each breed was collected directly from Portuguese Kennel Club archives. The number of potentially breeding females for each breed over time was calculated as the harmonic mean of breeding females since the first registries until the year 2001 (the majority of females only breed from the age of 2 up to 8 years old) because this is the least biased stationary estimator when comparing fluctuating, rapidly expanding or contracting, populations (Frankham and others 2002). These calculations are based on the Portuguese Kennel Club records only and mainly involve show dogs. Currently, these are the most accurate demographic data available due to a lack of a reliable census of working dogs for each breed.
Blood samples (2 ml) were taken into vacutainers containing ethylenediaminetetraacetic acid (EDTA) (10% w/v) and kept frozen until processed. Hairs, including the bulb, were plucked (up to 40–50 per individual) and kept dry. Tissue samples (ear biopsies) were preserved in dimethyl sulfoxide (DMSO)–salt buffer (20% DMSO, 0.25 M Na–EDTA, and NaCl to saturation, pH 8.0) at –20°C. DNA was extracted from whole blood and tissue according to standard proteinase K/Phenol–Chloroform protocols (Sambrook and Russel 2001), followed by an extra ethanol precipitation step, or using the Nucleospin Blood QuickPure kit (Macherey-Nagel, Düren, Germany) following the supplier's recommendations. DNA was extracted from hair bulbs in a Chelex 20% solution (protocol adapted from Walsh and others 1991).
DNA Amplification, Purification, and Sequencing
An mtDNA fragment of 887 bp, comprising a segment of the cytochrome b, the tRNA-Thr, the tRNA-Pro, and a segment of the control region, was amplified using a single pair of primers (all primers are numbered in relation to the complete mtDNA sequence determined by Kim and others 1998): L15210
[GenBank]
5'-ACA TGA ATT GGA GGA CAA CCA GT-3' (a shortened version of Shields and Kocher's [1991] L15774 primer) and H16097
[GenBank]
5'-TAT GTC CTG TGA CCA TTG ACT GA-3' (S. Funk, Institute of Zoology, London). L15210
[GenBank]
plus the following 5 internal primers were used for sequencing: H15377
[GenBank]
5'-TTT GAG TCT TAG GGA GGG CG-3' (designed by A. E. Pires), L15360
[GenBank]
(Hoelzel and others 1991), L15515
[GenBank]
5'-GTG TCA GTA TYT CCA GGT-3' (designed by A. E. Pires), L15805 (Southern and others 1988), and H16075
[GenBank]
5'-GCA CCT TGA TYT TAT GCG T-3' (designed by A. E. Pires).
Polymerase chain reaction (PCR) amplifications were performed in a 25-µl reaction volume with 100–200 ng of DNA from blood and tissue samples or 8–10 µl of extract from hair bulbs, as template, 1 U of Taq polymerase (Gibco [now Invitrogen], Paisley, UK), 1x reaction buffer (Gibco 10x stock contains 200 mM Tris–HCl pH 8.4 and 500 mM KCl), 1.5–2 mM MgCl2 (Invitrogen, Paisley, UK), 20 µM of each deoxynucleoside triphosphate (ABgene, Epsom, UK), 0.5 µM of each primer, and Sigma water. Nonacetylated bovine serum albumin (MBIFermentas, Vilnius, Lithuania) was included at a concentration of 0.024 µg/µl for amplification of blood-extracted DNA and 0.48 µg/µl for hair bulbs-extracted samples. Amplification reactions were performed in a GeneAmp PCR System 9700 (Perkin Elmer) thermal cycler, with an initial denaturation step of 94 °C for 3 min; followed by 40 cycles (60 cycles for hair samples) of denaturation at 94 °C (40 s), annealing at 55 °C (30 s), and extension at 72 °C (1 min); followed by a final extension step at 72 °C for 7min. For hair samples, we used a ramp between the annealing and extension steps at 50% of the maximum speed (programmable in the thermocycler) to make the temperature transition slower and increase the number of stable primer–template complexes undergoing extension in each cycle. A negative control was included in each set of amplifications. PCR products were separated on 1.5% agarose gels, cleaned using the GeneClean Turbo kit (Qbiogene, Carlsbad, CA), and eluted in 35 µl of elution solution. Purified products were sequenced in both directions using the ABIPrism BigDye Terminator Cycle Sequencing Ready Reaction Kit (version 2.0) and following the manufacturer's instructions. Products were separated on a semiautomated DNA analyzer (ABI 377) and sequences were edited, assembled, and aligned using the program SEQUENCHER 3.1.2 (Gene Codes Corporation) and submitted to GenBank using SEQUIN (http://www.ncbi.nlm.nih.gov/Sequin/).
Population and Phylogenetic Analyses
DNA Polymorphism
Arlequin version 2.0 (Schneider and others 2000) was used to compute indices of genetic diversity. Haplotype diversity H and nucleotide diversity 
were determined for each population. Pairwise genetic distances between haplotypes were calculated under the Tamura–Nei model (Tamura and Nei 1993), after excluding positions with insertion/deletions. Heterogeneity of substitution rates per site across the D-loop region was taken into account and gamma distribution parameters (Yang 1994) estimated with the best-fitting model of sequence evolution, as determined by MODELTEST version 3.06 (Posada and Crandall 1998). Pairwise FST values were estimated and significance at the 5% level determined with 10 000 permutations. Arlequin was also used to carry out an analysis of the molecular variance (AMOVA; Excoffier and others 1992). Three groups were defined taking into account the geographic origin of the samples: Portugal, Spain, and North Africa.
Sequences with no missing data were collapsed into haplotypes using the program COLLAPSE (Version 1.0), available at http://darwin.uvigo.es. All haplotypes were compared with the DNA sequence information stored in the GenBank database. This was conducted using the "basic local alignment search tool" (BLAST) program (Altschul and others 1990), available through the National Center for Biotechnology Information (Bethesda, MD). Chiperm version 1.2 (Posada 2000) was used to test for significant associations between haplotypes and breeds or dog populations using the algorithm developed by Hudson and others (1992). The significance of the 
2 statistics was approximated by Monte Carlo simulation (Roff and Bentzen 1989) by permuting the contingency tables; 100 000 permutations were performed.
Phylogenetic relationships among haplotypes (887 bp) were estimated using the neighbor-joining (NJ) method in PAUP version 4.0b10 (Swofford 2002). In order to analyze the sequences reported in this study in a wider context, 54 haplotypes of 554 bp (accession numbers: AF531664, AF531668, D83609, AF531670, AF531671, AF531672, AF531674, AF531675, D83606, AF531679, AF531682, AF531685, AF531687, AF531656, AF531693, AF531696, AF531700, AF531702, AF531710, AF531658, AF531731, AF531732, AF531733, AF531734, D83607, D83625, D83634, AB007402, AF531722, AF531723, AF531724, AF531725, AB007380, D83601, AF531728, AF531729, AF531730, AF531715, D83636, AF531717, AF531718, AF531719, AF531720, AF531721, AF531735, AF531736, AF531737, AF531738, AF531740, AF531739, AF531741, D83611, D83637, AB007381) were imported from GenBank to represent the 6 major dog clades described by Savolainen and others (2002). These haplotypes represent 20 of the 75 haplotypes described by Savolainen and others (2002) for clade A and all haplotypes described for clades B, C, D, E, and F. Haplotypes from wolves, golden jackal, and coyote were also included (accession numbers: AF008140, AF008137, AF008142, AF008135, AF008138, AF008139, AF008141, AF184048, AF008158). From the 3 Iberian wolf haplotypes described by Vilà and others 1997 (W1, W2, and W3), we selected W1 as a representative of wolves from Iberia. Among W1, W2, and W3, there are only 5 variable positions out of 261. In order to construct an NJ tree integrating sequences from the studies mentioned above, the 887-bp fragments generated in this study were trimmed to 554 bp. The best-fitting model of sequence evolution was determined by MODELTEST version 3.06 (Posada and Crandall 1998). The reliability of the nodes was assessed with 1 000 bootstrap iterations (Felsenstein 1985).
An intraspecific gene genealogy was inferred using the median-joining network algorithm (Bandelt and others 1999) in NETWORK, version 4.1, available at http://www.fluxus-engineering.com. Gaps were treated as evolutionary events, and data were analyzed with all characters weighing equally. The tolerance parameter 
was set to 0. Haplotype frequencies were converted into proportional areas in the graphical output.
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Results |
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Genetic Diversity and Differentiation
The primers L15210 [GenBank] /H16097 amplified a sequence comprising part of the cytochrome b gene (112 bp), the tRNA-Thr and tRNA-Pro genes (135 bp), and the hypervariable portion at the 5' end of the control region (640 bp). Forty-nine different haplotypes were obtained from 164 individuals (accession numbers: AY706476–524) (Figure 1). Forty-eight phylogenetically informative sites were found (5% of the total fragment), and, of these, only positions 4, 56, 145, 162, 206, and 225 are outside the control region. Changes corresponded to synonymous transitions. A strong transitional bias of 39.2:1 was observed, a common finding in the mtDNA of mammals (Graur and Li 2000). Due to indels, 3 sites contained gaps (positions 255, 722, and 729). Nucleotide frequencies for the entire fragment were A = 0.28420, C = 0.26180, G = 0.15250, T = 0.30150. The low guanine (G) content is a common result in vertebrate mtDNA (e.g., Tamura and Nei 1993).
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Of the 49 haplotypes found in this study, a BLAST search conducted against the sequences in GenBank revealed that 40 are new or unique haplotypes. Differences between the 40 new haplotypes and the dog sequences deposited in GenBank ranged from 1 to 15 nucleotides in 887 bp (0.1–1.7%). Twelve of the 49 haplotypes found in this study are shared among the 13 dog populations sampled, and the remaining 37 haplotypes are breed or dog population specific (Table 1). The total number of haplotypes found per breed or dog population (shared and nonshared) varies between 1 and 13 (Table 1). The number of nonshared haplotypes, that is, haplotypes that are only found in 1 of the 13 dog groups of this study varies between 0 and 7 (Table 1). Within the sampled individuals, 11 haplotypes, 4 from Aidi, 5 from Sloughi, and 2 from Tunisian dogs, were only found in dogs from North Africa (Table 1). With the exception of the Portuguese Warren hound, Aidi and Sloughi were the breeds showing the highest number of nonshared haplotypes (4 and 5, respectively, Table 1). Portuguese village dogs showed the highest value of haplotypic diversity (0.97 ± 0.04), followed by Aidi (Table 2). Among the remaining breeds, only the Portuguese Warren hound and Alentejo Shepherd dog showed haplotype diversities higher than 0.90. Haplotype diversity was not correlated to sample size (R2 = 0.0324, graph not shown). Among the endangered Portuguese native dogs, Portuguese Water dog had a relatively high haplotypic diversity (0.82 ± 0.08).
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Nucleotide diversity per site (
± SD) was on average high, ranging from 0.014 ± 0.007 (Estrela Mountain dog) to 0.002 ± 0.001 (Portuguese Pointer), with the exception of the Castro Laboreiro Watchdog that showed no diversity (Table 2). This statistic is not highly sensitive to sample size, as already noted by Vilà and others (1999). Castro Laboreiro Watchdog showed a single and private or breed-specific haplotype (H25) in the 14 analyzed animals, of which 11 were working dogs and 3 were show dogs. Estrela Mountain dog, Alentejo Shepherd dog, and Spanish mastiff showed similar values of genetic diversity. It is noteworthy that high levels of nucleotide diversity were also found in the Northern African dogs (Table 2).
Genetic differentiation among breeds and dog populations was statistically significant (2 = 1177.675, P < 0.001). Pairwise FST ranged from nearly 0 (between Aidi and Portuguese Village dogs) to 0.801 (between Castro Laboreiro Watchdog and Portuguese Pointer) (Table 2). Pairwise FST values between Portuguese village dogs and dogs from outside of Portugal (Spanish mastiff, Aidi, Sloughi, and Tunisia dogs) were near 0. Castro Laboreiro Watchdog and Portuguese Sheepdog were the only breeds with all pairwise FST values statistically significant. Castro Laboreiro Watchdog, Portuguese Sheepdog, Portuguese Pointer, and Portuguese Water dog, the least diverse breeds, showed the greatest genetic differentiation values from the Portuguese village dogs (significant at the 5% level), which represent the "background" mtDNA variation of dogs in Portugal. Within the livestock guarding dogs group (Castro Laboreiro Watchdog, Estrela Mountain dog, Alentejo Shepherd dog, Spanish mastiff, and Aidi), Castro Laboreiro Watchdog was the most isolated, with no mtDNA variation and significant FST values for all breed comparisons (
= 0.05) (all FST values 
0.396). Estrela Mountain dog and Alentejo Shepherd dog showed little genetic differentiation from each other. Spanish mastiff showed substantial differentiation from all Portuguese livestock guarding dogs, although to a much lesser extent from Estrela Mountain dog and Alentejo 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 differentiation from the Alentejo Shepherd dog (FST = 0.027 and not significant). Portuguese Warren hound showed significant FST values with the breeds Portuguese Sheepdog, Castro Laboreiro Watchdog, Portuguese Pointer, and Azores cattle dog. All FST values among Portuguese Warren hound subpopulations (varieties) were near 0 (data not shown). FST values between each breed and the dogs that compose the background genetic population (Portuguese village dogs) of their region ranged from –0.066 to 0.073, and none were significant (data not shown). AMOVA showed subdivision between breeds (
ST = 0.171, P < 0.0001), with around 80% of the variation found within populations (breeds). Variation 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.
Phylogenetics
The best-fitting model of sequence evolution determined by MODELTEST (hierarchical likelihood ratio test statistic) was the HKY85 + I + G model (Hasegawa and others 1985) with gamma distribution correction for rate heterogeneity among sites. The shape parameter of the distribution () and proportion of invariable sites (I) were 0.6306 and 0.8646, respectively. Only clades with bootstrap values equal or higher than 70% are considered here (see Hillis and Bull 1993).
Figure 2 shows an NJ unrooted phylogram of all haplotypes found in the samples of this study (887 bp). Mitochondrial lineages are not clustered by geographic origin or traditional breed classification: the same haplotypes occur in different populations and are scattered throughout different clades. Forty-nine percent of the haplotypes found in this study were in clade A and with all breeds represented, although with weak bootstrap support. Clades B, C, and D are supported by a high bootstrap value (83%) and include all breeds except Castro Laboreiro Watchdog (H25). Clade D, previously described as being regionally restricted to Turkey, Spain, and Scandinavia (Savolainen and others 2002), includes Estrela Mountain dog and Alentejo Shepherd dog haplotypes from Portugal (H38 and H40) and Aidi haplotypes from Morocco (H2 and H6) (AY706513 [GenBank] , AY706515 [GenBank] , AY706477 [GenBank] , and AY706481 [GenBank] respectively) (Figures 2 and 3). Few nodes showed significant bootstrap values, but most major partitions are well supported. When 54 domestic dog haplotypes from Savolainen and others (2002) and wild canid sequences from Vilà and others (1997) (fragments of 554 bp) were included, the structure of the NJ tree is very similar to that obtained by Savolainen and others (2002) (data not shown). We recovered all clades except E and F. Regarding the 554-bp sub–data set of the sequences generated in this study, we identified one new haplotype for clade D (AY706481 [GenBank] ) obtained from Morocco. For the other clades, 12 new haplotypes were found: in clade A, 5 (Portugal and Morocco, H19, H25, H41, H42, H44); clade B, 3 (Portugal and Morocco, H30, H34, H45); and clade C, 4 (Portugal and Spain, H16, H27, H29, H32) (AY706494 [GenBank] , AY706500 [GenBank] , AY706516 [GenBank] , AY706517 [GenBank] , AY706519 [GenBank] , AY706505 [GenBank] , AY706509 [GenBank] , AY706520 [GenBank] , AY706491 [GenBank] , AY706502 [GenBank] , AY706504 [GenBank] , AY706507 [GenBank] , respectively). The following haplotypes were breed or population-specific within this study: Castro Laboreiro Watchdog, 1 (H25); Estrela Mountain dog, 2 (H41, H42); Portuguese Warren hound, 4 (H29, H30, H32, H34); Spanish mastiff, 1 (H27), Aidi, 1 (H6); Sloughi, 2 (H44, H45) and Portuguese village dogs, 2 (H16, H19) (Table 1). The W1 Iberian wolf haplotype grouped with dog haplotypes from clade B, along with haplotypes from all breeds except Castro Laboreiro Watchdog, Portuguese Sheepdog, Aidi, and Portuguese Pointer. The C. aureus and C. latrans sequences were both highly distinct, as expected.
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The haplotype network (Figure 3) shows the same relationships as the NJ tree (Figure 2), but more detail is evident. Eight missing haplotypes (median vectors, mv) were detected. In clade A, all breeds are represented, but 4 median vectors are also included. The most frequent haplotypes, H7 and H10 (AY706482 [GenBank] and AY706485 [GenBank] ), respectively, are present in 16 (7 dog populations) and in 19 (6 dog populations) animals. These frequent haplotypes are connected to other haplotypes, from which they mostly differ by singletons. Haplotype H25 (AY706500 [GenBank] ) in clade A is only one mutational step (transition in position 422) different from haplotype H28 (AY706503 [GenBank] ). Haplotype H28 is shared by the Portuguese Pointer, Alentejo Shepherd dog, and Portuguese Warren hound breeds. Clade A is connected to Clade C by 11 mutational steps, including 2 median vectors. Clade C is connected to Clade B by the same number of steps. In the network, Clade D comprises 4 divergent lineages and is separated from the rest of the haplotypes by 8 steps. This clade comprises only 3 breeds in this study, whereas clades B and C represent 9 and 8 dog populations, respectively. Haplotypes H7, H10, H21, and H5 were shared between Portuguese and African breeds.
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Discussion |
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TopMethodsResultsDiscussionReferences |
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Genetic Diversity and Differentiation
Because most haplotype diversities were high (Table 2), we would expect the
2 test implemented in Chiperm to be powerful in detecting genetic differentiation between breeds or populations (Hudson and others 1992). Genetic differentiation was especially apparent due to the number of private haplotypes found in several breeds. The lack of differentiation between Portuguese village dogs and dogs from outside Portugal (Spanish mastiff, Aidi, Sloughi, and Tunisia dogs) is probably a consequence of the high diversity found in all these breeds and/or populations. Portuguese Warren hound subtypes (varieties) are not differentiated using mtDNA. Portuguese Water dog was listed in the Guinness Book of World Records in 1970 as the world's rarest dog breed (Molinari 1989) and has come close to extinction twice in the last century, with a total of only 50 animals in 1974 (Molinari 1993). However, the genetic diversity of Portuguese Water dog is surprisingly high. In Portuguese Kennel Club books, registrations for this breed have been less than 50 animals per year for 40 years and have never exceeded 450 animals per year. Although this breed is very popular in the United States, only one individual could be sampled from there (a dog imported from the United States and living in Portugal), and other Portuguese Water dogs living in Portugal share its haplotype (H11). Further efforts need to be undertaken to test more individuals from lineages exported to the United States from Portugal in the 1960s (Molinari 1993). Concerning Castro Laboreiro Watchdog, even though it is a livestock guarding dog like Estrela Mountain dog, Alentejo Shepherd dog, and Spanish mastiff, it was never involved in the large transhumance movements in which all other livestock guarding dog breeds participated (Geraldes 1996) and has been isolated for long periods. It was, instead, involved in small, geographically restricted migrations within a remote area, and contact with other dog populations is likely to have been scant. This is a possible explanation for its mtDNA uniqueness. Although working animals lacking the breed standard phenotype can be found in the area, these animals were not sampled for this study. However, they might be a possible addition to future analyses. Expected heterozygosity values inferred using microsatellites loci indicate that Castro Laboreiro Watchdog is one of the least diverse breeds in Portugal (Amaro 2001; AE Pires, F Simões, F Petrucci-Fonseca, and MW Bruford, in preparation). Within the dogs sampled for this study, position 422 shows a transition (A to G) that separates Castro Laboreiro Watchdog from all other dogs. Therefore, this nucleotide can provisionally be considered a breed-specific marker in the study area. As can be seen in the network (Figure 3), this haplotype differs by one base from a more common haplotype present in several dog breeds (Portuguese Pointer, Portuguese Warren hound, and Estrela Mountain dog). Probably Castro Laboreiro Watchdog as it is today, an mtDNA monomorphic dog breed, is the result of a severe bottleneck from a more diverse ancestral population of Castro Laboreiro Watchdogs. Considering all haplotypes from Savolainen and others (2002) and Vilà and others (1997), the guanine in position 422 is found in one dog from East Asia (isolate: A56, accession number: AF531707). However, the Castro Laboreiro Watchdog haplotype remains unique when compared with this East Asian dog due to a difference in another nucleotide position (position 430). Although less likely, this haplotype may represent a lineage tracing back to the livestock guarding dogs dispersal from Southwest Asia (associated with human migrations), where livestock was first domesticated (Mannion 1999).
The low levels of genetic differentiation between Estrela Mountain dog and Alentejo Shepherd dog are expected based on historical information: these breeds have a probable common origin, and until recently both have been in close contact due to the transhumance practice (Alpoim 1999). In contrast, genetic differentiation between Spanish mastiff and both Estrela Mountain dog and Alentejo Shepherd dog is more pronounced as genetic exchange that might have occurred during the long period of extensive transhumance is likely to have been more restricted. At the origin of the Azores cattle dog, the Portuguese native breeds Castro Laboreiro Watchdog, Estrela Mountain dog, and Alentejo Shepherd dog are considered founders (Amaral and Veiga 2004). Our results only support a major contribution by the Alentejo Shepherd dog because the FST value between the Azores cattle dog and the Alentejo Shepherd dog is the lowest value and is not significant.
It is interesting to note that livestock guarding and herding dog breeds, namely Estrela Mountain dog, Alentejo Shepherd dog, and Azores cattle dog, do not have a statistically significant divergent mtDNA composition from that of the "background" gene pool in the region where they are from, probably due to the high haplotypic diversity revealed by the Portuguese village dog population. Finally AMOVA results suggest that, although breeds share haplotypes, there is breed differentiation. Further sampling in Spain and North Africa may shed more light on the geographic structure of Iberian and North African dog breeds.
The high levels of diversity in African dogs could be explained by their antiquity and/or reported introgression. Both Aidi and Sloughi are considered rare (Miguil 1986; Ouar 1994). At the beginning of the 20th century, most of the dog population in the Atlas mountainous area comprised Aidi dogs. In contrast, by 1994, only 49% of the dog population corresponded to Aidi breed individuals, and in parallel with a reduction in numbers, there was documented introgression with hunting dogs (Ouar 1994). This breed may also be less intensively managed than many European breeds. The breed standard was described in 1969, but the majority of animals are free ranging for guarding purposes (Ouar 1994), making possible introgression with feral dogs and/or other dog breeds. The date of origin of the Sloughi remains speculative, but it is considered an ancient breed. Representatives of African Sighthound-like dogs date back to more than 7000 years (Giudicelli 1975). At present, it is mainly used to hunt desert hares and gazelles and indirectly to protect goat and sheep herds by hunting their predator, the golden jackal (Miguil 1986). A number of factors have contributed to its present low population size, including a decrease of hunting activities using dogs, the popularity of firearms, and a reduction in its geographic distribution (Miguil 1986). Finally, the high genetic diversity observed in Tunisian dogs is expected because the dogs sampled belong to the local village dog population from no specific breed. Tunisian dogs show considerably less differentiation from the other African dogs and from the Spanish mastiff than from the Portuguese native dog breeds. African domestic dogs were found to have distinct haplotypes and haplotype frequencies from Portuguese dogs, and although some haplotypes are shared, no south–north cline in frequency could be detected in our data as found in cattle, for which admixture was detected by Cymbron and others (1999).
Phylogenetic Analysis
The lack of an obvious underlying geographic or breed-related structure in the NJ tree could indicate that most of the breeds are derived from the same ancestral stock and retain many of the ancestral maternal haplotypes and/or that extensive introgression has occurred in the process of breed formation and development. Similar results were obtained by Tanabe and others (1991) based on blood proteins; Okumura and others (1996), Tsuda and others (1997), Vilà and others (1997), and Savolainen and others (2002) based on mtDNA control region; and Jordana and others (1999) using morphological and behavioral characteristics. Another explanation may be that most breeds have originated too recently, and 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 and others (2004) sampling purebred dogs from closed gene pools found that breeds could be genetically differentiated using biparentally inherited microsatellites markers. However, by sampling only 5 unrelated pedigreed animals from each breed, the authors did not carry out an extensive and representative sampling across each breed, thereby maximizing the probability of detecting a signal of breed structure.
Most of the haplotypes in this study clustered within the 3 major phylogenetic clades A, B, and C described in Savolainen and others (2002), implying that Iberian dogs are unlikely to be the result of an additional domestication event. All clades found by Savolainen and others (2002) were extensively represented in the phylogenetic analysis, although the proportion in clade A (26.6%) was reduced relative to their study. Therefore, the 8 breed-specific haplotypes found in Iberian and North African dogs for clades B, C, and D represent additional diversity in these clades. This unique diversity is potentially important information to the conservation of domestic dog genetic variation in this region.
The application of single-genetic locus surveys is not without problems (Avise 2004), and this work should be augmented by neutral nuclear DNA markers and genes associated with the phenotypes under selection. Currently, studies based on nuclear DNA markers such as amplified fragment length polymorphisms and autosomal microsatellites are ongoing (AE Pires, F Simões, F Petrucci-Fonseca, and MW Bruford, in preparation). MtDNA does not account for male-mediated gene flow, a dominant force in domestic animal evolution (Petit and others 2002). Male dog microsatellites loci from the nonrecombining portion of the canine Y chromosome are already available (Olivier and others 1999; Bannash and others 2005), and their analysis among breeds revealed a high breed and geographic structure (Bannash and others 2005). Similarly, it is likely that the study of the Y chromosome can bring additional detail to the phylogenetic analysis 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 association of DNA mutations, in particular intragenic tandem repeats in developmental genes, with phenotypic variability, allowed identification of a possible molecular mechanism responsible for morphological and functional variation. Neff and others (2004) by using a pharmacogenetic mutation as a molecular marker were able to unravel the emergence of formally recognized breeds. Another example is the work of Vilà and others (2005) who analyzed variation in genes from the major histocompatibility complex (MHC) potentially under balancing selection to infer past relationships between domestic mammals and their wild ancestors. The authors found high levels of genetic diversity suggesting that domestic mammals might have acquired much of their current genetic diversity by backcrossing several times with their wild ancestors. Typing nuclear genes from the MHC in each of the breeds found to contain unique mtDNA haplotypes in our study would probably reveal additional MHC alleles.
Implications for Conservation
Dog breeds should be viewed as dynamic populations, in which historic phenomena such as admixture, introgression, genetic isolation, and drift have occurred (Neff and others 2004). Portuguese dog breeds have experienced major demographic fluctuations over the past 20 generations, with a consistent decline through disuse of breeds in a working context, followed by a rapid expansion in many cases due to the fashion of showing dogs and for the pet trade. Concerning its mtDNA, Iberian and North African breeds show, in general, considerable diversity, including the possession of some newly described sequences. In this study that focused on marginal working dog breeds, several haplotypes emerge as novel, and conservation planning for these breeds should therefore take into account the unique genetic characters highlighted here. Following Weitzman's concept for livestock that involves the use of a "diversity function" (Bruford and others 2003), Castro Laboreiro Watchdog and Portuguese Sheepdog would become priorities for conservation on the basis of mtDNA alone. The considerable mtDNA diversity in the dogs of Iberia and North Africa prevents, with one exception, its use as a marker for breed designation, and these dog breeds should be studied with several other kinds of nuclear markers available to accurately assess breed distinctiveness.
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Acknowledgments |
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The research was supported by Fundação para a Ciência e a Tecnologia (grant PRAXIS XXI/BD/21677/99) and AGRO (project 31106). We thank Carla Cruz, Silvia Ribeiro, the dog owners, and breed clubs who contributed to this study; Margarida Gomes for helping collecting data from the Portuguese Kennel Club; Carlos Fernandes for advice in the laboratory and data analyses; Isabel Amorim, Carlos Fernandes, 2 anonymous reviewers, and the corresponding editor for helpful discussions and comments on the manuscript. We also thank the Portuguese Kennel Club for facilitating access to the database on dog registries.
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Footnotes |
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Corresponding Editor: Robert Wayne
Received August 26, 2005
Accepted May 4, 2006
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