Research Article |
Corresponding author: José Maurício Barbanti Duarte ( mauricio.barbanti@unesp.br ) Academic editor: Jesus Maldonado
© 2020 Analorena Cifuentes-Rincón, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Maurício Barbanti Duarte.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cifuentes-Rincón A, Morales-Donoso JA, Sandoval EDP, Tomazella IM, Mantellatto AMB, de Thoisy B, Duarte JMB (2020) Designation of a neotype for Mazama americana (Artiodactyla, Cervidae) reveals a cryptic new complex of brocket deer species. ZooKeys 958: 143-164. https://doi.org/10.3897/zookeys.958.50300
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Mazama americana (red brocket deer) is the genus-type species (first species described for this genus) and the basis for the identity of other Mazama species. Mazama americana is one of the most abundant and widely distributed deer species in the neotropical forest. However, recent studies suggest that this taxon belongs to a species complex. Our goal was to collect an animal at the type locality (topotype) in French Guiana with the aim of characterizing the morphological (biometric, craniometric), cytogenetic (Giemsa, C-banding, G-banding and NOR) and molecular (mitochondrial DNA) features. The comparisons showed that the collected specimen was very similar morphologically to specimens from other South American populations, but it was cytogenetically and molecularly very different from any of the cytotypes already described for this species, corroborating the existence of a complex of cryptic species. The data suggest that the M. americana topotype is a different species from all the cytotypes already described in the literature and which occupy the southern region of the Amazon River. The characterization and designation of the M. americana neotype is the first step toward a taxonomic reorganization of the genus Mazama, with the potential identification of new species.
biodiversity, chromosomes, cytogenetics, French Guiana, mitochondrial DNA, morphology, red brocket deer, topotype
The genus Mazama Rafinesque, 1817 (Mammalia, Artiodactyla, Cervidae) has 10 species widely distributed throughout the neotropical region (
Several cytogenetically studied species have shown an extensive intraspecific polymorphism (
These results have been corroborated by mitochondrial DNA variation, which clearly demonstrates the differences between some populations of M. americana Erxleben, 1777 (
These taxonomic uncertainties resulted in the species being categorized by the International Union for Conservation of Nature as “deficient data”, since the identity of the current taxon had no value as an evolutionary unit (
The species was described in 1777 by JCP Erxleben, a German naturalist, who is considered to be one of the founders of modern veterinary medicine. He did not collect any specimens; however, he used a series of reports by other authors as a basis for their description, mentioning the following characteristics: “M. rufo-fufeus, ore nigro, gula alba, auriculae longitudine quatuor pollicum, oculi magni nigri, nares magnae, oris regio nigra, crura poftica longiora anticis, cauda brevis, pili breues mollesque, capitis collique fupra fufci, colli fubtus albi, corporis crurumque rufofufci, vngulae nigrae, timidiffimus, celerrimus, agilis, natat per fluuios, caro bona”. Cayenne, in French Guiana, was considered by Erxleben as the type locality for M. americana.
The absence of the species holotype for a more complete morphological and genetic analysis calls for the need to describe a current topotype of the species and to propose a neotype based on it. Thus, in this work we propose a neotype for the species, as well as its morphological and genetic characterization, based on a male specimen collected in French Guiana, near the type locality of the species. In addition, we performed comparisons with the known populations of this species already studied (
An adult male specimen of M. americana (Fig.
Eighteen body measurements were taken using a digital caliper (0.05 mm precision) and a measuring tape. Based on these measurements, a statistical analysis of the quantitative data was performed, along with those of 41 M. americana individuals (adult males and females) and four individuals of different species used as an external group from the NUPECCE database through cluster analysis using the Paleontological Statistics, PAST 3.20 program (
Analysis of the external morphology of the specimen was performed based on criteria used by
Thirty-six cranial measurements were recorded using a digital caliper (0.01 mm accuracy), based on the criteria proposed by
Metaphase chromosome slides were prepared from tissue culture (
DNA was extracted from a muscle sample following the protocol of
The sense and antisense strands of all amplified fragments of two mitochondrial genes were sequenced. The two complementary strands were aligned, thus obtaining the consensus sequence from the Clustal W program (
The phylogenetic relationships between the different M. americana populations and the neotype were studied by Bayesian inference analysis (
Bayesian inference analyses were performed using 50,000,000 generations over four chains with two replications, adopting a 25% burn-in discard. To estimate the posterior probability, the Markov Chain Monte Carlo method was used. All trees were edited in the FigTree v. 1.4.0 program (
The collected animal is presented in Figure
Amended description of Mazama americana Erxleben, 1777 (Mammalia, Cervidae): Deer with predominant red coat, resembling reddish-brown brick color, gray in the face and neck region. The most intense color tone in the dorsal region. The side region the same tone as the back, slightly paler. The abdominal region the same color as the flanks, slightly brownish. Red tail in the dorsal region the same color as the back, and white in the ventral region. Whitish inguinal, buccal, gular and inner region of the ears. Black-colored posterior limbs in the caudal region, brown in the cranial region, external proximal region the same color as the lateral body region, white internal proximal region, brown external distal region, slightly reddish in the most distal portion, internal distal region also slightly brown, and the most distal portion slightly reddish. The lower and upper orbital bands slightly lighter than the rest of the face. Relatively deep lacrimal fossa. The outer ear surface lightly covered with brown hair. Smooth, varying hair lengths according to the body region, shorter and thinner hair covering the muzzle, the outer and inner surface of the ear (the latter partially hairless), the chin and the distal region of the anterior and posterior limbs. Somewhat longer hairs on the hips and tail. Strip of anteverted hair on dorsal midline of neck with more pigmented, blackened terminal band. Presence of a tuft of hair on the back of the head immediately preceding the horns. Relatively large and thick horns, dorso-caudal inclination, slightly curved and parallel to each other. Horns covered with soft tissue.
Recording location: Régina, French Guiana
Collection point coordinates: 4°19'52"N, 52°07'22"W
Specimen deposited in: Museum of Deer Research and Conservation Center (NUPECCE)–FCAV – São Paulo State University (UNESP) – Jaboticabal Campus.
Classification number: NPC079 (full skull, post skull, and taxidermized skin)
Tissue sample deposited in: JAGUARS collection, Kwata NGO, Cayenne, French Guiana, reference M3426_JAG
DNA sequence deposit numbers: MN726911 (Cyt-b), MN726914 (D-Loop)
Karyotype: 2n = 45 + 3Bs, FN = 51, sexual system XY1Y2
Synonymy: Given the cytogenetic and molecular results obtained to date, there is a high probability that several names currently synonymized with M. americana are valid names. Therefore, only names given to animals from French Guiana will be considered synonyms of M. americana, which are: Cervus rufus Cuvier, 1817 and Mazama pita Rafinesque, 1817.
The results of the cluster analysis made with body measurements did not reveal morphometric differences between the distinct geographic groups of M. americana. The distance tree (Fig.
Distance tree (cluster analysis) made with cranial measurements of different M. americana cytotypes, in parenthesis, found in Brazil (SA-Santarém, JU-Juína, RO-Rondônia, PR-Paraná, CA-Carajás) compared to M. americana neotype and other Mazama species (M. gouazoubira, M. nemorivaga and M. bororo).
The collected animal presented a cytogenetic constitution with diploid number (2n) of 45 chromosomes and 51 chromosomal arms (fundamental number, FN). The biometric analysis classified the pairs 1 and 2 belonging to Group A; 3, 4 and 5 to Group D; and 6 to 21 to Group E. The three chromosomes classified as supernumerary or B, were acrocentric and showed no numerical variation between the metaphases analyzed. The sexual system was XY1Y2, due to an X-autosomal fusion (Fig.
Under AgNOR staining, the telomeric regions of one of the chromosomes of pair 3 and two chromosomes of pair 6 were marked. The C-band (Fig.
The schematic representation of the G-banding of the M. americana neotype is shown in Figure
The tree generated by the analysis of the mitochondrial Cyt-b gene (Suppl. material
It is important to highlight that the species M. bororo and M. nana Hensel, 1872, taxonomically well-recognized and occurring in the south and southeast of Brazil, were grouped with M. americana from Rondônia and Juína. The distance between M. nana and M. bororo is smaller than the distance between several M. americana strains.
The analysis of the control region of the mitochondrial DNA D-Loop (Suppl. material
The concatenated tree of the genes Cyt-b and D-Loop (Fig.
Mazama americana Erxleben, 1777 is the name given to a species commonly recognized for its wide distribution in the Neotropics. However, Erxleben’s description was based solely on morphology, currently considered insufficient to discriminate species of the genus Mazama (
Thus, the absence of any specimen of the type series, the lack of critical information in the original description, as well as the large taxonomic uncertainties in this species complex (
The genus Mazama is characterized by rapid diversification and morphological parallelism (
The individuals belonging to the different cytotypes of M. americana found in Brazil (
In the tree of morphological distances, generated from the cranial measurements of the different M. americana cytotypes, we found two clades, where individuals belonging to the same cytotype are positioned in both clades, thus showing there is variation in individual cranial morphometric characteristics, which do not appear to have any geographical relation. Similarly,
The results obtained with our morphological and morphometric analyses reveal that there is morphological parallelism between M. americana variants, suggesting the existence of closely related characteristics, even in phylogenetically distant groups, due probably to recent diversification from their last common ancestor (
The chromosomal polymorphism found in red brocket deer is surprising and shows high levels of intra- and interspecific chromosome variation (
In the neotype chromosomes, the constitutive heterochromatin blocks are evident in chromosomes of Group A, weak in Group D and almost imperceptible in the first pair of Group E. According to
As previously reported by
Through the use of chromosomal banding techniques, important results were generated for the karyotype study of M. americana. Based on their comparison, it was observed that there is a chromosomal difference between the six M. americana cytotypes and the neotype due to pericentric inversions, tandem fusions and centric fusions, as previously reported by
The large chromosomal variation found in M. americana can be explained by the theory of chromosomal fragility proposed for Mazama (
Consequently, it is clear that the proposed neotype does not belong to the same species as the known M. americana cytotypes (
The result of the concatenated analysis of the two mtDNA fragments (Cyt-b and D-Loop), broadly followed the results obtained from the analysis of the separate genes, showing at least two evolutionary units for M. americana, yielding results very similar to previous studies (
The specimen of M. americana analyzed in this study complies with all the conditions required by the International Code of Zoological Nomenclature (1999) in force to be considered as neotype. The proposal of a M. americana neotype based on the detailed description of a current topotype opens great possibilities for describing new species within the genus Mazama. At this time, since there is a M. americana pattern (neotype), it is possible to define where the current cytotypes will be positioned. It is necessary that the entire nomenclature assigned to Mazama be reviewed from a cytogenetic point of view. For this, it would be necessary to access the chromosomal pattern for each of the available names. This can only be achieved if current topotypes are collected to define their karyotypes and position them taxonomically. In addition, molecular analysis from type series in museums could be used to help clarify the taxonomy (
It should be reiterated that it remains a formidable challenge to resolve the relationships between recently separated species, as in, for instance, the case of M. americana. However, this study has been able to confirm the existence of different species within the M. americana complex, as previously proposed by
This is the first reference available in the literature regarding the establishment of a neotype for M. americana, which is the starting point for the description of new species and possible change in the nomenclature of the genus Mazama.
We thank Coaraci who collected the animal for this study. Fundação de amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) provided financial support. We also thank João Airton Boer for his support for the laboratory work at the NUPECCE/UNESP. We thank Natália Aranha de Azevedo for the drawings of the animal heads used in the molecular tree.
Table S1. Samples used in this study for genetic analysis
Data type: molecular data
Explanation note: Nupecce’s identification, species, gene accessed, accession number in genbank, origin (locality) and source (when collected).
Table S2. Biometric data of the M. americana neotype
Data type: species data
Explanation note: Measured in cm and mass in kg.
Table S3. Cranial measurements of M. americana neotype represented in millimeters
Data type: species data
Figure S1. Phylogenetic tree of the Cyt-b gene
Data type: phylogenetic tree
Explanation note: Bayesian Inference (BI) Analysis. The values represent the posterior probability of the analysis. External group: R. tarandus, M. gouazoubira and O. bezoarticus.
Figure S2. Phylogenetic tree of the D-Loop gene
Data type: phylogenetic tree
Explanation note: Bayesian inference (BI) Analysis. The values represent the posterior probability of BI. External group: O. bezoarticus, R. tarandus, M. gouazoubira.