Anthropogenic threats have increasingly isolated the populations of Mazama temama (Erxleben, 1777) and limited the gene flow in this species. Knowledge of the phylogeographic structure of this species is therefore essential for its conservation. Thus, in this study, we describe the phylogeographic structure of two M. temama populations of Veracruz and Oaxaca, Mexico. We sequenced the D-Loop region of the mitochondrial DNA of 16 individuals, in order to estimate the diversity and genetic differentiation (F_ST), Tajima’s D index, "Mismatch distribution" test; a phylogram and a haplotype network was constructed and we performed multidimensional scaling analysis to test the hypothesis of association between geographic distance and genetic diversity. The haplotypic and nucleotide diversity was high, indicating divergent populations (F_ST = 0.223), while the Tajima’s D index (-1,03300; P > 0.10) determined disequilibrium in the D-Loop region, derived from a population expansion that was evidenced in the "Mismatch distribution" test and confirmed with the haplotype network in the form of a star. Four lineages were identified in the phylogram (Veracruz n = 3, Oaxaca n = 1), evidencing geographic and reproductive isolation between the two populations. This was confirmed by the multidimensional scaling analysis, which evidenced recent evolutionary divergence between the populations analyzed, which are considered evolutionary units of conservation.

Central American red brocket deer, lineages, nucleotide diversity, phylogram

The Central American Red Brocket, Mazama temama (Erxleben, 1777) is a deer species that has been targeted by illegal hunters for local commercial purposes or as a source of protein (Leopold 1959; Gallina and Mandujano 2009), as well as for its habitat (Gallina and Mandujano 2009; Bello-Gutiérrez et al. 2010). Even so, this deer species is considered Data Deficient by IUCN (Bello et al. 2016). This category is due to uncertainties about the threats that the species has suffered (Bello et al. 2016). Although threats such as those reported above are known, the degree of danger posed to the species is not clear (Bello et al. 2016). Thus, basic information about the species is essential to support conservation strategies.

Since certain populations of M. temama are found isolated in remnants of vegetation, it is important to study the phylogeography of this species in order to understand the micro-evolutionary processes these populations have undergone historically.

Phylogeography is a multidiscipline that has been used as a management tool for the conservation of species, and involves the study of micro and macroevolutionary processes that govern the geographic structure of genealogical lineages of species (Avise et al. 1987; Arbogast and Kenagy 2001; Vázquez-Domínguez 2002; Vázquez-Domínguez 2007) and features two important components: demographic structure and genetic structure (Avise 1992). An approach in the phylogeographic context can thus help to clarify changes in genealogical lineages based on their geographic distribution (Avise and Hamrick 1996; Avise 2000).

On the American continent, particularly in Mexico, phylogeography has been used to study populations of wild fauna, especially endemic mammals, or those sought by hunters, or those included in some category of risk, leading to important implications for their conservation (Vázquez-Domínguez 2005; Vega et al. 2007; Vázquez-Domínguez et al. 2009; Escobedo-Morales et al. 2016). There is evidence that phylogeographic studies have had a considerable impact on species management (Vázquez-Domínguez and Vega 2002), especially in the ungulates, describing cervid lineages in order to define particular strategies of management (Serna-Lagunes et al. 2021), identifying bottlenecks and low gene flow in species of interest to hunters (Castillo-Rodríguez et al. 2020) and evidence of genetic divergence among lineages of the same species (Fuentes-Hurtado et al. 2011). In this sense, in this study we describe the phylogeographic structure of two populations of M. temama.

The analysis of the nucleotide composition of the obtained sequences indicated a higher average frequency of Cytosine bases in the M. temama population of Veracruz, while the Oaxaca population presented a greater proportion of Adenine bases (Table 2).

The two populations of M. temama showed differences in genetic diversity. In the population of Oaxaca, there was a lower number of polymorphic sites compared to the population of Veracruz, which explains why the number of haplotypes was different. That is, each sample analyzed was found to be an independent haplotype, and haplotypes were not shared between the populations. Haplotypic and nucleotide diversity was high in both populations, indicating that the sampled individuals of each population were heterogeneous in terms of the D-loop region (Table 3). This is consistent with the results of the index of genetic differentiation (F_ST) = 0.223, which indicated genetic differences between the populations of Oaxaca and Veracruz, as a result of not sharing haplotypes (Hi = 0).

The Tajima’s D index was negative (-1,03300; P > 0.10), indicating the presence of deletions or mutations, such that the populations were not found in neutral equilibrium. The "Mismatch distribution" test fitted to the model of a population of constant size (Oaxaca: R² = 0.500, initial theta (θ)= 14.750, tau (τ)= 48; Veracruz: R² = 0.217, initial theta (θ)= 14.75, tau (τ)= 82.571), evidencing a possible population expansion by the variation shown in the graphs of this test (Fig. 2).

The phylogram [(consistency index (CI)= 0.631579 and, retention index (RI)= 0.427673)], showed paraphyletic relationships between the samples, indicating that there is no geographic structure. For example, some Veracruz samples were closer to Oaxaca samples than other Veracruz populations (Fig. 3).

The haplotype network presented a structure in the form of a star (Fig. 4), with 351 total non-segregating sites and 233 mutations. This is commonly observed in populations that have undergone a process of demographic expansion and was consistent with the results of the Tajima’s D index and the "Mismatch distribution" test.

According to the MDS (Fig. 5), the M. temama individuals most related according to nucleotide distance were those found in southern Veracruz and Oaxaca (upper right extreme of the dimensional plane), while the least related were those of central and southern Veracruz (extreme lower left of the dimensional plane), which explains the genetic relationship based on geographic distance; the stress value was good (0.14), with a value of R² = 0.87, indicating a significant relationship between the pairs of sequences of the matrix of nucleotide and geographic distances (Fig. 5). Finally, the PCA determined that the greatest proportion of the variance was accommodated in the first two principal components (>80%). Principal component 1 explained 63.8 of the variances (Table 5), while principal component 2 explained 16.3% and together these components explained 80.3% of the total accumulated variation (Fig. 6).

Figure 2. 

"Mismatch distribution" for the M. temama populations from Oaxaca (left) and Veracruz (right), in Mexico.

Figure 3. 

Phylogram of genealogical relationships between the two M. temama populations of Oaxaca and Veracruz, in Mexico. Values on the branches represent the fit of the Bootstrap test.

Figure 4. 

Haplotype network of the two M. temama populations of Oaxaca and Veracruz, in Mexico.

Figure 5. 

Multidimensional scaling analysis applied to the matrix of nucleotide and geographic distances of pairs of M. temama sequences.

Figure 6. 

Principal components analysis applied to the matrix of nucleotide and geographic distances of pairs of M. temama sequences.

In this study we performed our first phylogeographic analysis on the species M. temama. Our results show that the populations analyzed underwent a genetic bottleneck and rapid population expansion.

Although we have studied a small sample of the population, these populations studied here do not share haplotypes due to the geographical distance between the two populations. On the other hand, it is possible that the reproductive behavior of this cervid means that the frequency of mtDNA transfer decreases, due to the philopatry of the females (Purdue et al. 2000; Purdue et al. 2006). It is only the males that migrate great distances to mate and thus the lineages of the females are presented in greater proportion with proximity to their area of birth (Nelson and Mech 1992). The lack of shared haplotypes between the two studied M. temama populations is evidence of the geographic and reproductive isolation.

The results of the "Mismatch distribution" test show an evident multimodal form, reflecting processes of sudden population expansion associated with a stochastic process of lineage extinction through genetic drift, recorded in the genetic structure of the populations (Rogers and Harpending 1992). This may reflect the threats that the species suffers in Mexico; M. temama is of hunting interest and subject to constant pressures of exploitation, habitat fragmentation, poaching, auto-consumption by local communities and predation by carnivores (Gallina 2005). This has led to a reduction in the effective population size where only certain lineages pass to the next generation, while others are extinguished or are present in lower frequency in the population. These results are to be expected considering that M. temama is a species that suffers from poaching, since a similar phenomenon has been reported in the cervid Pantholops hodgsonii in China, which is at risk of extinction due to the loss of genetic diversity (Xiang-Dong et al. 2005).

The populations analyzed have different evolutionary histories, as shown by the string haplotypic and nucleotide relationship between the populations, evidencing differentiated lineages between the two populations (Grant and Bowen 1998). It is concerning that the populations studied present low values of gene flow, as occurs in some other species of Neotropical cervids. This is associated with the fact that M. temama inhabits fragmented ecosystems with no biological corridors to allow migration of individuals for the purposes of mating. Phylogeographic studies must be conducted in more populations of M. temama in order to evaluate their status of genetic diversity and to generate a program of conservation of their diversity, since their ecological niche is restricted to vulnerable ecosystems due to the reduction and fragmentation of their habitat (Serna-Lagunes et al. 2014).

The authors offer their sincere gratitude to the reviewers of our manuscript, whose observations and comments enabled the improvement of this research. Thanks go to Linda Elvira Rodríguez Vera, Jorge Noriega Loyo and the Área de Biología Molecular of the Laboratorio Cordobés de Diagnóstico Pecuario for validation of the PCR and provision of materials and equipment. Thanks also to the UMA El Pochote and the Colección Nacional de Mastozoología (CIIDIR), for the tissue samples. To the project “Characterization of animal genetic resources of the high mountains, Veracruz: application of phylogeography and ecological modeling (PRODEP: 511-6/18-9245/PTC-896) for their support in finalizing this publication.