Assessing the Taxonomic Status of the Gray Brocket Mazama simplicicornis argentina Lönnberg, 1919 (Artiodactyla: Cervidae)

© 2023 Academia Sinica, Taiwan Open Access Assessing the Taxonomic Status of the Gray Brocket Mazama simplicicornis argentina Lönnberg, 1919 (Artiodactyla: Cervidae) Eluzai Dinai Pinto Sandoval1, Gabrielle Queiroz Vacari1, Juan Pablo Juliá2, Susana González3, Miluse Vozdova4, Halina Cernohorska4, Svatava Kubickova4, Daniela C. Kalthoff5, and José Mauricio Barbanti Duarte1,* 1Núcleo de Pesquisa e Conservação de Cervídeos (NUPECCE), Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista (UNESP), Jaboticabal-SP, Brazil. *Correspondence: E-mail: [email protected] (Duarte) E-mail: [email protected] (Sandoval); [email protected] (Vacari) 2Facultad de Ciencias Naturales. Universidad Nacional de Tucumán, Argentina. E-mail: [email protected] (Juliá) 3Departamento de Biodiversidad y Genética, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. E-mail: [email protected] (González) 4Veterinary Research Institute, Brno, Czech Republic. E-mail: [email protected] (Vozdova); [email protected] (Cernohorska); [email protected] (Kubickova) 5Swedish Museum of Natural History, Frescativägen 40, Stockholm, Sweden. E-mail: [email protected] (Kalthoff) Received 27 October 2022 / Accepted 28 April 2023 / Published 12 July 2023 Communicated by Jian-Nan Liu Mazama simplicicornis argentina is the name that was given to describe a gray brocket collected by Lönberg in 1919 in the central Chaco region of Argentina. Subsequent authors, based on morphological similarities, considered this name to be a synonym for the species Subulo gouazoubira Fischer, 1814 from Paraguay. In the absence of genetic analyses to compare the Argentinian and Paraguayan gray brockets, we aimed to clarify the taxonomy of M. simplicicornis argentina through an integrative assessment using morphological, cytogenetical, and molecular data from its holotype and a current topotype. Qualitative skull features and cranio-morphometric results of M. simplicicornis argentina showed a great similarity with the S. gouazoubira neotype characters. The diploid chromosome number of M. simplicicornis argentina topotype corresponded with the karyotypical pattern of S. gouazoubira with 2n = 70 and FN = 70, showing a great similarity in all classic and molecular cytogenetic results and revealing the homologies between karyotypes. The phylogenetic analysis of mitochondrial genes used in this study (concatenated partial ND5 and Cytb gene) allocated the M. simplicicornis argentina specimens in the monophyletic clade of S. gouazoubira with a branch value of 100%. These results show that there is no discontinuity between the Argentinian and Paraguayan gray brockets. Therefore, the individuals originally described as M. simplicicornis argentina should be recognized as S. gouazoubira. Key words: Animal cytogenetic, Cervidae, Gray brocket, Mitochondrial DNA, Morphology Citation: Sandoval EDP, Vacari GQ, Juliá JP, González S, Vozdova M, Cernohorska M, Kubickova S, Kalthoff DC, Duarte JMB. 2023. Assessing the taxonomic status of the gray brocket Mazama simplicicornis argentina Lönnberg, 1919 (Artiodactyla: Cervidae). Zool Stud 62:30. doi:10.6620/ ZS.2023.62-30. BACKGROUND The gray brocket deer Subulo gouazoubira (G. Fischer 1814) is a small to medium-sized deer that is distributed from south of the Amazon region to Uruguay and the north of Argentina (Black-Décima et al. 2010). The species was first described by Azara (1801) who identified individuals from Paraguay and named them Gouazoubira, a name of Guarani origin, that was later invalidated for not following the Principle of Binomial Nomenclature (ICZN 1999). Based on Azara’s observations, the species was named Cervus Zoological Studies 62:30 (2023) doi:10.6620/ZS.2023.62-30 1 © 2023 Academia Sinica, Taiwan gouazoupira by G. Fischer (1814) and subsequently transferred to the genus Mazama by Rafinesque (1817), due to the morphological similarities with the species that compose the genus. Rather recently, the spelling gouazoubira has been defined as the appropriate citation for the species (ICZN 2001: Opinion 1985), and the name Mazama gouazoubira was adopted by many authors for this taxon (Cabrera 1960; Rossi 2000; Black-Décima et al. 2010). However, later studies identified these similarities as adaptive convergence and thus contradicting the monophyly of the genus Mazama (Gilbert et al. 2006; Duarte et al. 2008; Gutiérrez et al. 2017; Heckeberg 2020). Authors have discussed this polyphyletic condition of the genus Mazama, in which the species are recovered in two subtribes, the Blastocerina and Odocoileina (Heckeberg 2020). In this sense, Azara’s individuals would be recovered within the Blastocerina subtribe, separate from M. americana (Erxleben 1777), the type species of the genus Mazama, that is recovered within the Odocoileina subtribe (Gutiérrez et al. 2017; Heckeberg 2020; Bernegossi et al. 2022). A recent study of the complete mitochondrial phylogeny confirmed this relationship and reviewed the applicability of the name-group Subulo Smith, 1827, to describe the gray brocket deer, resulting in the renaming of Subulo gouazoubira (G. Fischer 1814) (Bernegossi et al. 2022). This nomenclature rearrangement was proposed as a starting point to assist in the taxonomic clarification of Neotropical deer, allowing for the comparison with other taxa described in the past that were categorized as synonym or subspecies by many authors (Bernegossi et al. 2022). This is the case of Mazama simplicicornis, an objective synonym attributed to Azara’s (1801) gray brockets by Illiger (1815), which was considered the valid name by many authors (Smith 1827; Lesson 1842; Gray 1850; Pelzeln 1883; Allen 1915; Lydekker 1915; Lönnberg 1919; Miranda-Ribeiro 1919). Lönnberg (1919) suggested that two subspecies were present in Argentina, M. simplicicornis gouazoubira (G. Fischer 1814) and a new one, which he named M. simplicicornis argentina based on an adult female collected in the Argentine Chaco. Lönnberg (1919) noticed that in comparison with Mazama simplicicornis from Paraguay, his new subspecies from Argentina had smaller skull and body dimensions, with the presence of a white spot below the nostril and behind the rhinarium, which was considered absent in the individual from Paraguay (Lönnberg 1919). During the 20th century, all the gray forms of brockets were considered to belong to M. gouazoubira, and for this reason, both subspecies of Mazama simplicicornis were synonymized with the M. gouazoubira (Cabrera 1960). Thus, Lönnberg’s gray brocket was renamed M. gouazoubira argentina and identified as a subspecies restricted to the locality Rio de Oro in the Central Chaco Region of Argentina. On the other hand, M. gouazoubira gouazoubira is distributed in the southern region of Brazil, the Pantanal, Paraguay, Uruguay and northern Argentina (in Tucumán, Santiago del Estero and Entre Rios Provinces) (Avila-Pires 1959; Pinder and Leeuwenberg 1997; Richard and Juliá 2001; Caraballo 2009; Periago and Leynaud 2009). The definition at a sub-specific level has been discussed by several authors with divergent positions. Some have defended the absence of morphological differences between S. gouazoubira subspecies and Azara’s description as the only gray brocket present in Argentina (Cabrera 1960; Czernay 1987). In this context, cytogenetic data is an important tool to clarify taxonomic status. This was demonstrated in studies analyzing subspecies of Mazama americana, which identified karyotipic divergneces that confirmed the validation of M. jucunda Thomas 1913; M. temama (Kerr 1792) and M. rufa (Illiger 1815) (Jorge and Bernishke 1977; Duarte and Jorge 2003; Peres et al. 2021; Sandoval et al. 2022). The revalidation of new species of Mazama confirms the importance of reviewing each taxon described and also raises doubts about the identity of other descriptions of species or subspecies that were considered synonyms of the former M. gouazoubira, now named S. gouazoubira. Thus, the goal of this study is to reassess the taxonomic status of Lönnberg’s (1919) Mazama simplicicornis argentina and clarify its relationship to the gray brockets of Paraguay. This will be realized with the assistance of morphometric as well as molecular (mtDNA) and cytogenetic methods. Lönnberg’s type specimen (female) and the topotype (male), both from Chaco Central, Argentina, are central in our analysis. MATERIALS AND METHODS Specimens and samples We collected an adult male topotype in Presidencia de la Plaza locality, in the Chaco Province of Argentina. The specimen received an identification number NUPECCE (T450) and catalog number (NPC172) at the Deer Research and Conservation Center (NUPECCE) museum where it is deposited. In addition, data and tissue samples of the M. simplicicornis argentina holotype (adult female) were collected in the Swedish Museum of Natural History at Stockholm (NRMMA620394) for craniometrical and molecular analysis. page 2 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan Morphological characterization After collecting, the topotype was photographed, and the skin was removed and treated with a tanning solution to preserve it. Aspects of general coat color, color of the neck, dorsal line of the body, tail, ventral region of the body, front and hind feet, pigmentation patterns in the hair of different regions of the body, length of hair in different regions of the body, occurrence of strips of anteverted hair and rounded hair tufts in the tarsal region were examined. Additionally, the facial color pattern was analyzed (Hershkovitz 1982). Different angles of the skulls were photographed to complement the documentation and description of the specimens. Following the criteria of von den Driesch (1976), we took 26 skull measurements presented in the collected topotype and holotype of M. simplicicornis argentina, and also, we measured adult individuals of S. gouazoubira, Mazama nemorivaga, Mazama americana and Mazama rufa species from the NUPECCE database (Table S2). The following were measured: total length, condylobasal length, basal length, short skull length, premolar 1 – prosthion, viscerocranium length, greatest length of the nasals, short lateral facial length, oral palatal length, lateral length of the premaxilla, length of the cheektooth row, length of the molar row, length of the premolar row, greatest inner length of the orbit, greatest inner height of the orbit, greatest mastoid breadth, greatest breadth of the occipital condyles, greatest breadth at the bases of the paraoccipital, greatest breadth of the foramen magnum, least frontal breadth, greatest breadth across the orbits, least breadth between the orbits, zygomatic breadth, greatest breadth across the nasals, greatest breadth across the premaxillae, and basion (defined as the highest point of the superior nuchal crest). We used the “Paleontological Statistics” PAST program (Hammer et al. 2001) to perform a cluster analysis by the Unweighted Pair Group Method with Arithmetic mean method (UPGMA) based on Euclidean distance of 26 skull measurements. The dataset was formed by male and female individuals to include both the topotype (male) and the holotype (female) of M. simplicicornis argentina. Cytogenetic analysis After collecting the topotype, 5 × 2 cm skin fragments from the inguinal region were collected and preserved in liquid nitrogen as described by Duarte et al. (2021). Then, the chromosomes were obtained by fibroblast in vitro culture according to Verma and Babu (1995). The chromosomal preparations were subjected to conventional Giemsa staining, G-banding (Seabright 1971, modified), C-banding (Sumner 1972), and Ag-NOR staining (Howell and Black 1980). Chromosomes were numbered according to the S. gouazoubira karyotype described in Bernegossi et al. 2022 showing correspondence to cattle (Bos taurus, BTA) chromosomes. Fluorescence in situ hybridization using BAC probes was performed to characterize the homologies between the karyotype of Mazama simplicicornis argentina topotype and the chromosomes of S. gouazoubira (2n = 70 and NF = 70). BAC clones were selected from the CHORI-240 cattle library based on the NCBI ARS-UCD1.2 Assembly data was obtained from BACPAC Genomics, Emeryville, CA, USA (Table S3). For DNA extraction, we used a protocol adapted from the method included in Wizard® Plus SV Minipreps DNA Purification Systems. BAC DNA was labeled with Green-DdUTP (Abbott, IL, USA), biotin 16-dUTP or digoxigenin-11-dUTP (Roche, Mannheim, Germany) using BioPrime® Array CGH Genomic Labeling (Invitrogen, Carlsbad, CA, USA). FISH was performed as described in Vozdova et al. (2019). A Zeiss Axio Imager Z2 (Carl Zeiss Microimaging GmbH, Jena, Germany) fluorescence microscope, equipped with appropriate fluorescence filters for the visualization of FISH results, was used. DNA extraction, amplification and sequencing The genomic DNA was extracted from the liver of the topotype using a modified protocol based on the methodology described by Sambrook et al. (1989), and from nasal bone of the holotype using a modified protocol based on the methodology described by Medrano et al. (1990) with optimizations performed as in González (1997). The samples were quantified by spectrophotometry and analyzed by agarose gel analysis, and then diluted in a solution for use. For the topotype, the amplification was performed using the protocol described by Peres et al. (2021) of the following partial genes: cytochrome b (Cytb 480 bp and 660 bp) (Hassanin et al. 1998; Duarte et al. 2008) and NAD5 Dehydrogenase subunit 5 (ND5 691 bp and 688 bp) (Caparroz et al. 2015). For the holotype, the amplification was performed with real time PCR using a Rotor Gene (Qiagen) and the following partial genes: cytochrome b (Cytb 224 pb) (González et al. 2009), NAD5 Dehydrogenase subunit 5 (ND5 224 pb) and NAD5 (ND5 251 pb) (Leandro 2019). The reaction was composed of 5 µL of SensiFASTTM HRM Kit (1X), 0.6 µL of each primer (10 µM), 5 µL of DNA (10 ng/µL) and 3.8 µL of water. For DNA amplification the cycles were 95°C for page 3 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan 2 minutes / 95°C for 5 seconds/ 54°C for 10 seconds (20 cycles) / 53°C for 10 seconds (20 cycles) / 52°C for 10 seconds (20 cycles) / 72°C for 20 seconds. Purification of the PCR products was performed with the Wizard SV gel and PCR Clean-Up System kit (Promega TM), followed by sequencing using the ABI BigDye Terminator kit (Applied Biosystems) in an ABI 3130xl automatic sequencer (Applied Biosystems). Phylogenetic analysis We generated mtDNA sequences of the holotype (Cytb 224 bp partial gene, ND5 224bp partial gene and ND5 249 bp partial gene) and topotype (Cytb 480 bp partial gene, ND5 691 pb partial gene and ND5 688 bp partial gene) (Table 1). From GenBank, we obtained available sequences of the Neotropical deer species M. americana sensu lato, M. rufa, M. nemorivaga, M. nana, M. pandora, M. jucunda, M. temama, S. gouazoubira, H. antisensis, B. dichotomus and O. bezoarticus to complement the matrix for phylogenetic analysis using the species A. alces as outgroup. The concatenated nucleotide sequences were aligned using the Clustal X program (Thompson et al. 1997), and the ends were manually replaced with BioEdit “N” (Hall 1999). The evolutionary model was generated with Modeltest 3.7 (Posada and Crandall 1998). Bayesian inference (BI) analyses were performed using the MrBayes 3 software (Huelsenbeck and Ronquist 2001), with 10,000,000 generations until obtaining a variance of < 0.01, adopting a 25% burnin discard. To estimate the posterior probability, the “Markov Chain Monte Carlo” (MCMC) method was used with nchains = 4, nruns = 2 and burninfrac = 0.25 for all genes. The tree obtained was edited with the FigTree v.1.4.0 software (Rambaut 2012). RESULTS The Mazama simplicicornis argentina Lönnberg, 1919, topotype shows a general gray color of body, tinged yellowish laterally, with presence of mental and nasal whitish patch, a brown pale mandibular Table 1. Mitochondrial gene sequences used for phylogenetic analysis of Mazama simplicicornis argentina Lönnberg, 1919, topotype and other neotropical cervids Species Cytb ND5 Origin Description Mazama simplicicornis argentina (NRM-MA620394) OP627522 OP627524 OP627525 Chaco Province, Argentina. Lönnberg 1919 This study Mazama simplicicornis argentina (T450) OP627521 OP627523 Chaco Province, Presidencia de la Plaza. Argentina This study Subulo gouazoubira (T377) MZ350858 MZ350858 Puerto Galileo, Paraguay Bernegossi et al. 2022 Subulo gouazoubira (T389) MZ350866 MZ350866 Puerto Arecutacuá, Paraguay Bernegossi et al. 2022 Subulo gouazoubira (T082) MZ350862 MZ350862 Camobi-RS, Brazil Bernegossi et al. 2022 Subulo gouazoubira KJ772514 KJ772514 Pantanal, Brazil Caparroz et al. 2015 Subulo gouazoubira (MRGsp2) JN632658 JN632658 Colombia Hassanin et al. 2012 Mazama nemorivaga (T24) MZ350861 MZ350861 Porto Velho-RO, Brazil Bernegossi et al. 2022 Mazama nemorivaga (T346) MZ350867 MZ350867 Lupar-PA, Brazil Bernegossi et al. 2022 Mazama nemorivaga JN632659 JN632659 Peru Hassanin et al. 2012 Mazama nemorivaga JN632660 JN632660 French Guiana Hassanin et al. 2012 Mazama americana (MAZ9472) JN632656 JN632656 French Guiana Hassanin et al. 2012 Mazama americana (T358) MZ350857 MZ350857 Cayenne, French Guiana Bernegossi et al. 2022 Mazama americana (T255) MN726909 MZ488890 Juina, Brazil Cifuentes-Rincón et al. 2020/ Peres et al. 2021 Mazama americana (T253) MZ350856 MZ350856 Juina, Brazil Bernegossi et al. 2022 Mazama rufa (T385) MZ488852 MZ488894 Foz de Iguaçu, Paraná, Brazil Peres et al. 2021 Mazama nana (T107) MZ350863 MZ350863 Paraguay Bernegossi et al. 2022 Mazama jucunda (T071) DQ789231 MZ488899 Paraná, Brazil Duarte et al. 2008/ Peres et al. 2021 Mazama jucunda (T215) MZ350859 MZ350859 P. E. Intervales-SP, Brazil Bernegossi et al. 2022 Mazama temama (T366) MZ350864 MZ350864 Veracruz, México Bernegossi et al. 2022 Hippocamelus antisensis JN632646 JN632646 Argentina Hassanin et al. 2012 Alces alces MF784602 MF784602 Poland Świsłocka et al. 2020 Ozotoceros. Bezoarticus (FNMA40) MZ350860 MZ350860 Brazil Bernegossi et al. 2022 Blastocerus dichotomus (CV21) OM543539 OM543540 Brazil Sandoval et al. 2022 page 4 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan band and a dark brown rostral stripe toward the antler button. The supra and infraorbital band has a pale yellow with brown-speckled hair, with presence of a white superciliary spot, and a frontal hair tuft. The specimen also shows big ears with inner border white, yellow hair laterally on the lower border, and white posterobasal auricular patch. The color of the dorsal neck to the anterior region of the body has gray-brown color and the posterior region has a dark brown color speckled with golden hairs. Flanks showed a pale brown color where the coat is longer with the presence of the yellowish hair. The perianal region and lower surface of the tail has a white color, the external proximal area of the members of the same color as the dorsum of the neck and the distal part shows a dark golden color (Fig. 1). The skull of the topotype and holotype showed a flat lacrimal fossa, two lacrimal foramina externally at the orbital border, separated from each other, with an extended vomerine septum, and a small, flat tympanic bulla. In addition, the topotype’s skull presented a short, thick, inclined pedicles and a preorbital region with inverted triangular shape (Fig. 2). All these characters were in accordance with the skull described for S. gouazoubira neotype, excepting the pedicles that in this last specimen was slender, but also short and inclined. The skull measures of these specimens were very similar within each other. The only difference was the length of nasal that in M. simplicicornis argentina topotype showed a greater size than the holotype described by Lönnberg (1919) and also, the S. gouazoubira (G. Fischer 1814) neotype. The cluster analysis of 26 craniometrical characters (Fig. 3) shows that the two individuals of M. simplicicornis argentina fall within the group of small brockets with S. gouazoubira and M. nemorivaga separated from a second group with M. americana and M. rufa. All S. gouazoubira specimens from different origins in Brazil, Paraguay and Argentina, formed a mixed group, in which both topotype and holotype were grouped in a subgroup with one individual S. gouazoubira from Santa Catarina state in Brazil. Cytogenetic description The cytogenetic analysis performed in the M. simplicicornis argentina (MSA) topotype revealed a karyotype with diploid number 2n = 70, XY, and absence of B chromosomes (Fig. 4). All autosomes and the X chromosome were acrocentric, showing correspondence in G-banding patterns and FISH results to S. gouazoubira chromosomes (SGO) (Fig. 4, Table S4). The Cbanding revealed heterochromatic positions in the centromeric regions of each chromosome. The NORs were detected in the telomeric regions of /MSA1 and MSA2 pairs. The BAC probe derived from the centromeric region of BTA1 (BAC106N15) hybridized in the centromeric region of chromosome MSA16, while the BAC probes derived from the proximal (BAC69G2), distal (BAC109I18) and telomeric (BAC273F5) regions of BTA1 hybridized to the chromosome MSA4 (Fig. 4). BAC clones derived from the BTA X hybridized in the proximal (BAC316D2X and BAC159O16) and middle (BAC 40H2) regions, and distally in the PAR region (BACs 453C5, 326C13) of the MSAX chromosome showing correspondence to the SGOX chromosome (Fig. 4 and Fig. S1). Phylogenetic analysis The Bayesian Inference tree of concatenated fragments of Cytb and ND5 showed two major clades. The first clade consisted of the Odocoileina subtribe, which included the species M. jucunda, M. nana, M. temama, M. rufa, and the paraphyletic M. americana. The second clade comprised the Blastocerina subtribe, encompassing the species S. gouazoubira, M. nemorivaga, H. antisensis, B. dichotomus, and O. bezoarticus (Fig. 5). Both the holotype and topotype of M. simplicicorins argentina were recovered in the Blastocerina subtribe, within the S. gouazoubira clade (100% branch value) with H. antisensis as sister taxon (90% branch value). The holotype was recovered in a subclade with individuals of S. gouazoubira from Paraguay and the topotype was recovered in a subclade with one individual from Brazil. In the same Blastocerina subtribe, M. nemorivaga and B. dichotomus were allocated as sister taxa. The species O. bezoarticus was shown as the first group that diverged in the Blastocerina subtribe (Fig. 5). Fig. 1. Lateral view of the adult male topotype of Mazama simplicicornis argentina (Lönnberg 1919) collected in the Chaco Province of Argentina. page 5 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan Fig. 3. Cluster analysis of skull measurements of Mazama simplicicornis argentina (MSA) (Lönnberg 1919) compared with Subulo gouazoubira, Mazama nemorivaga, Mazama americana and Mazama rufa; star = collected adult male topotype; arrow = adult female holotype. Fig. 2. Skull views of Mazama simplicicornis argentina (I) Holotype and (II) Topotype, compared with (III) the skull of Subulo gouazoubira neotype (Bernegossi et al. 2022, adapted). A = right lateral view, B = left lateral view, C = dorsal view and D = ventral view. page 6 of 11 Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan DISCUSSION Qualitative comparison between M. simplicicornis argentina individuals and the S. gouazoubira neotype showed a great morphological similarity of all characters. The color pattern of external body features of amended description of S. gouazoubira neotype were present in M. simplicicornis argentina topotype. The topotype shared the white spot behind the rhinarium and below the nostril with the holotype that Lönnberg (1919) considered absent in the animal from Paraguay, however, this character has been reported as an intraFig. 5. Bayesian Inference of concatenated mitochondrial genes Cytb (partial 224 bp) and ND5 (partial 224 bp and partial 249 bp) of neotropical deer species including Mazama simplicicornis argentina topotype (star) and holotype (arrow). Outgroup = A. alces. Fig. 4. Karyotype of a male topotype of Mazama simplicicornis argentina (MSA) (Lönnberg 1919) collected in the Chaco Province of Argentina. From left to the right: chromosomes under C-banding, G-banding and ideogram of Subulo gouazoubira (SGO) showing correspondence to MSA chromosomes (adapted from Bernegossi et al. 2022). The NOR positions are marked with asterisks. FISH results using BAC probes derived from BTA1 and BTAX are presented on the right of the SGO4. page 7 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan specific variation within S. gouazoubira populations (Allen 1915; Rossi 2000). As there is no study that correlates the rostral spot with geographic delimitation, this feature should not be considered as diagnostic of Lönnberg’s Argentinian gray brocket M. simplicicornis argentina. Lönnberg (1919) also noticed a size difference between the M. simplicicornis argentina holotype and the Azara’s (1801) gray brocket from Paraguay, however, all morphological measurement in the topotype of M. simplicicornis argentina were in the pattern described for S. gouazoubira species (González et al. 2018; Bernegossi et al. 2022). This size variation observed by Lönnberg (1919) could be also associated with the wide intraspecific variability that has been reported among S. gouazoubira populations (Duarte and Jorge 1998; Rossi 2000; Black-Décima et al. 2010). In addition, the cluster analysis of skull measurement features revealed that the M. simplicicornis argentina topotype can be allocated in the same small brocket group as S. gouazoubira individuals. It is important to note that M. nemorivaga individuals were also recovered in the same group, which contrasts with previous studies that found them to be morphometrically different from S. gouazoubira due to smaller skull measurements (Gonzáles et al. 2019; Rossi 2000). These diverging results may be related to the different sampling numbers of M. nemorivaga that were assessed in each analysis. Qualitative characters such as larger, rounded ears, smaller eyes, smaller orbital cavities, and wider auditory bulla have been cited as efficient in distinguishing S. gouazoubira from M. nemorivaga with larger orbital cavities, and narrow auditory bulla (Rossi 2000). Although the skull measurement distance failed to separate the M. simplicicornis argentina topotype from M. nemorivaga, the similarity of qualitative characters with S. gouazoubira is the main morphological correspondence between M. simplicicornis argentina and S. gouazoubira. This study contributed the first cytogenetic description of a gray brocket from Chaco Region in Argentina. The diploid number of the M. simplicicornis argentina topotype corresponds to the S. gouazoubira pattern with 2n = 70 and FN = 70 (Neitzel 1987; Tomazella 2016; Valeri et al. 2018; Bernegossi et al. 2022). The descriptions of individuals with 2n = 69 and FN = 70, as in the case of the S. gouazoubira neotype, were associated with the occurrence of intraspecific chromosomal polymorphism commonly described for the species characterized by heterozygous centric fusion arrangement (Valeri et al. 2018; Bernegossi et al. 2022). However, we did not observe this or any other translocation in any of the analyzed metaphases of the M. simplicicornis argentina topotype. In addition, the B chromosomes were not observed in the metaphases of the topotype, which are commonly reported in S. gouazoubira individuals. The descriptions had shown an intra and inter individual variation of B chromosome numbers from 0 to 5 (Tomazella 2016). Our comparative FISH results with the bovine BAC clones revealed X chromosome structure similar to the acrocentric variant present in S. gouazoubira (Bernegossi et al. 2022) and other species from subfamily Cervinae (Frohlich et al. 2017). Also, the FISH positions of the BAC clones for BTA1 corresponded to their localization in S. gouazoubira karyotype, with the BTA1 centromeric region in one separated acrocentric chromosome (MSA16, SGO16) and the proximal, distal and telomeric part of BTA1 in another chromosome (MSA4, SGO4) (Bernegossi et al. 2022). Therefore, the great similarity of Ag-Nor staining, Cand G-banding with the S. gouazoubira described ideogram, and also the identical BAC clone’s hybridization in M. simplicicornis argentina individual and S. gouazoubira are evidence of the homologies between karyotypes of these species (Bernegossi et al. 2022). We inferred that there is no chromosomal difference that could represent a reproductive barrier between Argentina and Paraguay gray brocket, as there is between other Mazama species (Galindo et al. 2021). The phylogenetic analysis of mitochondrial markers used in this study (concatenated partial ND5 and Cytb gene) allocated the M. simplicicornis argentina topotype and holotype in the monophyletic clade of S. gouazoubira with a branch value of 100%. The recovery of individuals M. simplicicornis argentina in separated subclades associated with S. gouazoubira from different origins apparently indicates an absence of genetic structure. However, the low branch values do not support this recognition. The phylogenetic results also confirmed the S. gouazoubira clade to be within the Blastocerina subtribe as previously described (Gutiérrez et al. 2017; Heckeberg 2020), further supporting the species as sister taxa of Hippocamelus antisensis, separated from the M. nemorivaga, Ozotoceros bezoarticus and Blastocerus dichotomus species. Finally, our morphologic, cytogenetic and phylogenetic analyses do not support M. simplicicornis argentina as a valid species or subspecies. Furthermore, we did not detect differentiation between the M. simplicicornis argentina holotype, topotype and S. gouazoubira neotype, and the other S. gouazoubira specimens. In conclusion, our analysis supports the idea that M. simplicicornis argentina should be considered as junior synonym of Subulo gouazoubira. This confirmation, in addition to revealing genetic data of gray brocket individuals from Chaco Argentina, page 8 of 11Zoological Studies 62:30 (2023) © 2023 Academia Sinica, Taiwan contributes to the taxonomic clarification of the Lönnberg’s (1919) description in the context of the resolution of the genus Mazama. Acknowledgments: We thank Subsecretaría del Ambiente y Biodiversidad de la Provincia del Chaco for the authorization and official permission to collect the topotype. We thank Daniel Charters (John Moores University, Liverpool) for assistance with images of the holotype specimen. We also thank João Airton Boer for his support with the laboratory work at the NUPECCE/ UNESP, Valdir Nogueira-Neto and Pedro Henrique Peres for assistance in the field trip to collect the topotype. This research was support by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) process n° 2017/07014-8 and 2019/06940-1, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code n° 001 and National Research Council (CNPq) process number 302368/2018-3. Authors’ contributions: EDPS contributed with the writing and performed all morphological and genetic analyzes of the specimens. GQV and SG contributed with molecular genetics analysis of the holotype. DK contributed with sampling and morphometric analysis of the holotype. MV, HC and SK contributed with cytogenetic analysis of the topotype, JPJ contributed with topotype’s collection and analysis of this study and JMBD organized the research conception and the article design and conducted the final revision of the article. Competing interests: The authors declare there are no competing interests. Availability of data and materials: The datasets presented in this study can be found in GenBank. 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