Intrapopulation Genetic Variability
Variation in the number of polymorphic alleles observed in the genetic parameter analysis (Table 3) may be associated with farm management (such as selecting a specific trait/phenotype or other breeding strategies) in sampling locations. Consequently, this may result in different alleles being fixed in these populations, yet all showed similar heterozygosity values. This indicates gene flow between the populations, since the number of polymorphic SNPs does not necessarily reflect the heterozygosity rates observed. Sampling sizes for the locations were different, which may confer bias to the analysis since a higher number of polymorphic markers were observed in populations with a higher number of individuals sampled.
Other studies calculating average heterozygosity for Pantaneiro horse populations using microsatellite (Giacomoni et al., 2008; Sereno et al., 2008; Cortés et al., 2017) and RAPD-PCR markers (Egito et al., 2007) were similar between each other but differed from our study. These earlier studies used multiallelic markers, explaining the differences with those found in the present study with SNPs. There are no published studies regarding the assessment of genetic diversity employing the use of SNP markers for the Pantaneiro horse breed.
Interpopulation Genetic Variability
Despite being significant, all FST values observed were low (Table 4). This low genetic distance suggests the existence of gene flow between populations, indicating that the natural environmental barriers imposed by the Pantanal ecosystem (especially flooded terrains) do not prevent reproduction between individuals from different locations. This is possible due to the breed's adaptative traits from inhabiting this biome, conferring these animals remain in the flooded area for long periods without experiencing foot-rot, for example (Ribeiro et al., 2008; Santos et al., 2008). This hypothesis is reinforced by the PCA results (Fig. 1), where animals from all populations overlapped with no clear segregation between them, indicating a lack of genetic structure. The Mantel test showed no significant correlation between populations genetic (FST) and geographical (km) distances. Some populations that were closer geographically had higher FST values than some that were further apart. Therefore, no reproductive isolation caused by geographical distance was discerned in the analyzed populations, which supports the hypothesis that the terrain does not hinder the gene flow between them.
Similar results for Pantaneiro breed populations were found by Giacomoni et al. (2008), showing low genetic distances varying from FST = 0.008 to 0.064. Authors also observed low inbreeding levels for these populations and suggest that combined with the breed's adaptative traits that allow for gene flow between animals from different locations, variation in FST results may be associated with local breeding strategies from each farm. Cortés et al. (2017) also observed low values of inbreeding for the breed. These results corroborate the absence of genetic differentiation observed for populations of the breed in the present study. McManus et al. (2013) observed considerable genetic variability for the breed between farms, but low between Pantanal counties, as well as low inbreeding values, corroborating the hypothesis above.
Population Genetic Structure (Admixture)
Genetic structure analysis (ADMIXTURE), together with the cross-validation test performed for the election of an ideal number of clusters (K), suggest the existence of four substructured populations (Fig. 3). However, only one population (Barra do Bugres) showed a somewhat higher number of animals with "pure" genetic structure. In addition, the major genetic component present in Barra do Bugres animals can be found in other populations, so it is not exclusive. This corroborates, once again, with the existence of gene flow between locations. According to McManus et al. (2013), the demand for famous stallions is high, accounting for movement between regions and farms. Moving from K = 3 to K = 4 a new structure appears, which is more intense in the Nhumirim population. Except for these two somewhat structured populations, those remaining (Campo Grande, Cuiabá, Poconé e Promissão) showed high levels of admixture. Therefore, it was not possible to observe four substructured populations.
More clusters (K = 7) made it possible to observe certain levels of differentiation in two other populations: Cuiabá and Promissão. The genetic components here can, however, also be found in all other populations to some extent. Adding more clusters (K = 9 and 11), this continues to be accentuated and three larger genetic structures can be observed: Barra do Bugres (with a majorly "pure" composition), Cuiabá and Promissão (somewhat "pure" portions, with a more intense presence of admixture). With the addition of more clusters, populations Campo Grande, Nhumirim and Poconé contained a high degree of admixture. Nhumirim animals, however, showed a genetic structure mostly exclusive to this population in K = 9 and 11 (Figure S2).
Sereno et al. (2008), with microsatellite markers, also observed intense admixture in the genetic composition of Pantaneiro horses, corroborating the high genetic diversity found in the present study, as well as the lack of genetic structure between the studied populations.
We raise the hypothesis that the selection of Pantaneiro horses may be using morphological characteristics as selection criteria instead of the animals' geographic location. This would explain the appearance of new substructured Pantaneiro populations with favourable alleles for selection based on breeding of animals with desired traits by determined breeders. The Pantaneiro breed may also have changed due to indiscriminate crossing with other breeds in the past (Santos et al., 2003) not sampled in this study, as indicated by the genetic contributions that are not present in other locally adapted Brazilian breeds (Bchara, 2021). However, the Pantaneiro breed presents alleles to be confirmed as a distinct breed compared to other naturalized Brazilian horse breeds. It was not possible, in this study, to determine the cause of the apparent substructures and admixture observed in the Pantaneiro horse breed. Nevertheless, McManus et al. (2008) selection should ensure that the breed does not lose its adaptive traits