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PHYLOGENETIC SIGNAL OF SMALL MTDNA AMPLICONS TO STUDY THE MAZAMA AMERICANA COMPLEX THROUGH NONINVASIVE GENETIC SAMPLING.
The red brocket deer (Mazama americana Exrleben, 1777) is considered a complex of cryptic species. The karyotype variation within the species is striking and results in a reproductive barrier that could lead to the isolation of divergent populations. Phylogenetic studies with mitochondrial genes evidenced the species division into two paraphyletic lineages and confirmed the isolation and monophyly of the main described cytotypes. Biological material evaluated to date is limited, given the low accuracy of its geographical origin and spatial gaps such as the lack of Cerrado and Pantanal samples. The difficulty to access forest deer species has been successfully overcome through non-invasive genetic sampling using scat-detection dogs to find feces in the wild. The design of appropriate genetic markers is essential for fecal DNA analysis, which is characterized by low quantity and fragmentation. Thus, the objective of the present work was to select small regions of mitochondrial DNA capable of amplification in degraded samples and analyze their phylogenetic signal in the context of the genetic variants already identified within the M. americana complex. We selected a set of 20 M. americana animals with cytogenetic information from NUPECCE biological collection to compose a reference matrix containing the main cytotypes described for the species and the neotype specimen. To this matrix, we added another nine specimens to represent the Odocoileini tribe as the ingroup (two M. americana, two M. bororo, one M. temama, one Odocoileus virginianus, one Odocoileus hemionus, one Mazama gouazoubira, one Ozotocerus bezoarticus) and four specimens of other Cervidae tribes to compose the outgroup of the analysis. We partially sequenced the Cyt B, Dloop, and ND5 genes of Mazama to form a 2032 bp reference matrix, while the other taxa had Its sequences downloaded from GenBank. From this reference matrix, we selected five small regions between 221 bp and 316 bp (two Cit B, two Dloop and one ND5) and designed specific primers for the M. americana complex. This set constituted a smaller matrix with 1151 bp, which represents 56% of the reference matrix size. We compared the polymorphism of both matrices by observing the number of variable sites and parsimoniously informative characters (PICs). The phylogenetic signal was compared by observing the clades topology and support in trees generated by Bayesian Inference. The small sequence matrix had 59% of polymorphic sites, and 62% of PICs relative to the reference matrix and the phylogenetic hypotheses were mostly congruent and well supported in both matrices. The difference between them was that the reference matrix recovered all cytotypes in monophyletic groups while the smaller matrix grouped two of them in a mixed clade. The primers and amplicons designed have the potential to be applied in non-invasive samples, which will provide an extensive, well delineated and with geographic assigned genetic sampling. This approach will help to understand the distribution and isolation of the red brocket deer genetic variants which, in turn, will provide necessary information for their conservation status assessment.
fecal DNA; karyotype variation; Bayesian Inference; Odocoileini, Cervidae.
FAPESP Processos 2017/07014 e 2017/02200-8
Pedro Henrique Faria Peres, Eluzai Dinai Pinto Sandoval, Márcio Leite Oliveira, José Maurício Barbanti Duarte