Genetic variability in maned wolf based on heterologous short-tandem repeat markers from domestic dog

INTRODUCTION

The maned wolf, Chrysocyon brachyurus, listed as near threatened with extinction by IUCN and vulnerable by IBAMA, is the largest South America canid (Dietz, 1984). Its geographic range covers the natural savannahs of central Brazil (the Cerrado), the north of Bolivia, Paraguay, the north of Argentina, the northwest of Uruguay, and a small area in the east of Peru (Langguth, 1975; Dietz, 1984; Moreira et al., 1998).

The main threats to maned wolf conservation are habitat loss and fragmentation due to agricultural expansion, road kills, susceptibility to disease transferred from domestic dogs, and the belief that maned wolves prey on domestic stock, resulting in their persecution (Dietz, 1984; Vieira, 1996; Rodrigues, 2002).

Microsatellites are widely regarded as the most versatile genetic markers yet discovered, and their applicability for identifying relationships in natural populations is well documented (Bruford and Wayne, 1993; Queller et al., 1993). However, some of the obstacles in the use of microsatellites are the time required, costs and difficulty in isolating these short-tandem repeats and their flanking regions from the genome of target organisms. An alternative approach to de novo development is to exploit the available information by cross-species amplifications among a range of phylogenetically related species (Moore et al., 1991; Blanquer-Maumont and Crouau-Roy, 1995; Pepin et al., 1995; FitzSimmons et al., 1995; Wintero and Fredholm, 1995; Coltman et al., 1996; Scribner et al.,1996; Gemmell et al., 1997; Hille, 2002).

The objective of the present study was to investigate the genetic diversity of the maned wolf based on microsatellite markers transferred from dog (Canis familiaris).

MATERIAL AND METHODS

Blood samples were collected from a hundred captive wolves from 11 Brazilian zoos (Brasília, Goiânia, Uberlândia, Uberaba, Araxá, Belo Horizonte, Varginha, Bauru, Sorocaba, São Paulo, and Curitiba) over two different periods (1994 and 1996) and also from 12 free-living individuals from the “Águas Emendadas Ecological Station” (ESECAE), in 1997.

Only the animals of known origin, born in the wild and not closely related, were used for genetic variability analysis, totaling 69 individuals. These individuals were gathered into four groups according to their capture region (not their zoo residence) and also taking into account existing landscape barriers such as rivers and mountain ranges. The four groups formed were considered here as the Brazilian region populations to be compared (Figure 1).

Approximately 10 mL of whole blood (with EDTA) was collected from each animal and transported to the genetics laboratory at the University of Brasília for appropriate processing. DNA extraction was performed with GFXTM “Genomic Blood DNA Purification Kit” from Amersham Pharmacia Biotech Inc. The procedures adopted were those described in the product’s manual.

Five tetranucleotide loci, originally isolated from a domestic dog genomic library (Francisco et al., 1996), were tested against the maned wolf in a cross-species amplification experiment. Table 1 shows the main characteristics of these markers for the species for which they were originally developed.

Amplification reactions were performed for each marker separately. The PCR reactions were carried out in a 25-µL final reaction mixture containing PCR buffer (1X), 250 ng of target DNA, 0.25 mM of each dNTP, 2 mM MgCl₂, and 1 unit Taq DNA polymerase (Biotools). Primer concentration was optimized for each marker: 0.8 µM (FH2088 and FH2054) and 0.96 µM (FH2006, FH2010 and FH2144). Samples were amplified in a PT-100 MJ thermocycler (Research, Inc.) by denaturing at 94°C for 1 min, followed by 29 cycles of denaturation at 94°C for 1 min, annealing at 56°, 58° or 60°C for 45 s and extension at 74°C for 1 min. The final extension step was for 5 min at 74°C.

For polymorphism analysis, 4 µL PCR reaction product was briefly mixed with 4 µL formamide loading dye and denatured at 95°C for 10 min, placed immediately in an ice-water bath, and then electrophoresed through 10%, 29:1 (acrylamide:bis-acrylamide) denaturing gels containing urea. Gel electrophoresis was performed at room temperature, in an appropriate vertical cube. A constant voltage of 150 V was used. The electrophoresis time was specific for each system, varying according to the size of the amplified fragments: 4 h for FH2088 and FH2054, 4 h and 30 min for FH2006, 6 h and 30 min for FH2010, and 7 h for FH2144. Bands were visualized by specific-staining procedures (Sanguinetti et al., 1994).

A 25-bp molecular weight marker (Invitrogen) was run adjacent to the control and wolf samples on a 10%, 29:1 (acrylamide:bis-acrylamide) non-denaturing gel to provide an estimated allele size of the product amplification.

Dog control samples were utilized in order to detect successful homologous loci. The majority of the amplifications displayed “stutter” bands typical of microsatellite loci.

Allele frequencies and heterozygosity values were calculated with Biosys-1 version 1.7 software package (Swofford and Selander, 1981). Polymorphism information content was estimated using the Cervus 2.0 program (Marshall et al., 1998).

RESULTS AND DISCUSSION

All five microsatellite loci amplified for the maned wolf samples. Despite the amplification success (100%), one locus (FH2144) was monomorphic (20%). All maned wolf allele sizes were smaller than the ones observed for the dog control sample. The sizes of the amplification products were 100-125 bp for the locus FH2088; 175 bp for FH2054; 225-250 bp for FH2006; 250-275 bp for FH2010, and 350-375 bp for FH2144.

The chance of a successful cross-species (heterologous) amplification of any DNA sequence is inversely related to the evolutionary distance between the two species as has been demonstrated in several other papers. Indeed, several studies have shown that microsatellites isolated from various species amplify the corresponding and polymorphic loci in closely related species but not in more distant ones (Moore et al., 1991; Primmer et al., 1996; Gemmell et al., 1997; Galbusera et al., 2000; Hille et al., 2002; Williamson et al., 2002; Gonela, 2003; Nguyen et al., 2005). In the present study, highly polymorphic tetranucleotide microsatellites, characterized and developed for Canis familiaris, were successfully transferred to Chrysocyon brachyurus (Table 1). Microsatellite transferability was also obtained for other carnivore species evolutionarily related to (Primmer et al., 1996; Gemmell et al.,1997; Koskinen and Bredbacka, 2000; Altet et al., 2001; Williamson et al., 2002) and very recently to maned wolf for different markers than the ones discussed here (Rodrigues et al., 2006). Genetic variability measures and allele frequencies for each microsatellite locus for the total population and for the different Brazilian region population samples are shown in Table 2. The whole population genotypic frequencies, as well as that of each regional population, were in Hardy-Weinberg equilibrium.

The number of alleles per locus for each regional population ranged from 3.50 (Triângulo Mineiro) to 4.25 (DF/GO), H_E from 66% (DF/GO and Belo Horizonte) to 68% (Triângulo Mineiro) and polymorphism information content from 56% (Belo Horizonte) to 59% (São Paulo) (Table 2). There were no genetic differences among regional population samples except for DF/GO and São Paulo (c² = 22.779; P = 0.04; Table 3).

The analysis of each locus revealed a significant difference for FH2088 locus (c² = 13.041; P = 0.04; Table 4).

Another study employing 14 isozyme loci for the same maned wolf samples, detected 42.9% polymorphism and an average heterozygosity of 5.6% (Moreira et al., 1998), which was similar to the results obtained for other canids (Francisco et al., 1996; Koskinen and Bredbacka, 2000; Altet et al., 2001). Nevertheless, Rodrigues (2002) detected extremely low levels of protein polymorphism (10%) for the population of maned wolves from ESECAE, which is a small conservation unit situated in the northern part of Distrito Federal. The observed heterozygosity values in the latter study were low (1.4%), when compared to other canids.

Another genetic assay involved 20 protein loci in 28 maned wolves from an area in northeastern São Paulo State. Although the area was divided into three sub-areas, considering the Mogi-Guaçu and Pardo Rivers as possible barriers to gene flow, the polymorphism and heterozygosity levels found were similar to those observed by Moreira et al. (1998) for the maned wolf and by other authors for other species of free-living canids, and the samples of each sub-area and the total sample exhibited genotype frequencies consistent with the genetic equilibrium model showing no evidence of inbreeding, nor population subdivision (De Mattos et al., 2004).

Although the results obtained for isozymes and microsatellite markers showed a considerable genetic diversity in maned wolf populations (except the one studied by Rodrigues, 2002), mtDNA markers suggest that this species shows the lowest mtDNA diversity (P = 0.0026), of all the carnivores already investigated (Prates Jr et al., 2004).

Considering the analysis for the four polymorphic loci in the total maned wolf population, the mean allele number (A) was 4.3 (± 0.5) and the mean expected heterozygosity (H_E), 0.669 (± 0.026). These results are in accordance with those observed for Lycaon pictus (A = 3.9; H_E = 0.61) (Girman et al., 2001), Canis lupus (A = 4.5; H_E = 0.62) (Roy et al., 1994), Canis rufus (A = 5.3; H_E = 0.55), and Canis mesomelas (A = 5.0; H_E = 0.67) (Wayne, 1996). Canis simensis, probably the most endangered canid, with fewer than 500 individuals remaining in small and highly isolated populations restricted to Ethiopian highlands, showed lower values for the number of alleles and heterozygosity (A = 2.4; H_E = 0.24; Gottelli et al., 1994) than those found for the maned wolf. It is important to note that three of the five species shown above are considered threatened by IUCN (C. rufus, C. simensis and L. pictus) (Sillero-Zubiri et al., 2004), while the other two are protected by the U.S. government (Wayne, 1996).

The genetic variability results for this maned wolf sample, which is considered threatened with extinction, did not show any loss of variability when compared to studies of other canids (Francisco et al., 1996; Koskinen and Bredbacka, 2000; Altet et al., 2001).

IMPLICATION FOR CONSERVATION

The results obtained should be considered with caution, since they reflect the genetic variability of the maned wolf in a sample that was collected ten years ago. Despite the fact that no significant reduction of the genetic variability for the species was found, it does not mean that this process is not underway. The present study represents a base of comparison for future evaluations of the conservation status of the species. The insularization process of Cerrado areas has accelerated in the last years, and if effective conservation measures are not soon adopted, future evaluations could reveal a severe threat to the species’ survival. The results obtained in the present study open a new perspective in conservation genetic studies of the maned wolf. The use of a larger sample in the analysis of new short-tandem repeat markers may not only provide greater robustness to the results obtained, but also assist in the elaboration and execution of strategies in conservation programs and management of the maned wolf, such as those developed by Pró-Carnivoros and the AZB (Brazilian Zoo Association).

ACKNOWLEDGMENTS

We are especially grateful to Dulce Maria Sucena da Rocha, Jader Marinho and Eucleia Contel for their constructive comments on this manuscript. Research supported by Conselho Nacional de Pesquisas (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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