A peer-reviewed open-access journal Zookeys 958: 143—164 (2020) SR Senos #ZooKeys https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research Designation of a neotype for Mazama americana (Artiodactyla, Cervidae) reveals a cryptic new complex of brocket deer species Analorena Cifuentes-Rincén', Jorge Alfonso Morales-Donoso', Eluzai Dinai Pinto Sandoval', Iara Maluf Tomazella', Aline Meira Bonfim Mantellatto*, Benoit de Thoisy’, José Mauricio Barbanti Duarte' I Nicleo de Pesquisa e Conservacdo de Cervideos (NUPECCE), Faculdade de Ciéncias Agrarias e Veterindrias da Universidade Estadual Paulista (UNESP), Via de Acesso Paulo Donato Castellane, s/n CEP: 14884—900, Jaboticabal-SP Brazil 2 Universidade Federal do Sul da Bahia, Campus Sosigenes Costa, Porto Seguro, BA, CEP: 45810-000, Brazil 3 Kwata NGO, 16 Avenue Pasteur, 97300 Cayenne, French Guiana Corresponding author: José Mauricio Barbanti Duarte (mauricio.barbanti@unesp.br) Academic editor: Jesus Maldonado | Received 20 January 2020 | Accepted 18 May 2020 | Published 11 August 2020 http://zoobank.org/5 1 CECFA3-BAD3-4791-845D-963F CB4127FA Citation: Cifuentes-Rincén 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 Abstract 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 compari- sons 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 designa- tion of the M. americana neotype is the first step toward a taxonomic reorganization of the genus Mazama, with the potential identification of new species. Copyright Analorena C. Rincon et al. 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. 144 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) Keywords biodiversity, chromosomes, cytogenetics, French Guiana, mitochondrial DNA, morphology, red brocket deer, topotype Introduction The genus Mazama Rafinesque, 1817 (Mammalia, Artiodactyla, Cervidae) has 10 spe- cies widely distributed throughout the neotropical region (Merino and Rossi 2010). However, there are controversies about the number of species and subspecies that com- pose the genus. Early taxonomic revisions suggested the existence of 17 species and 11 subspecies (Allen 1915), four species and 10 subspecies (Cabrera 1960), and six spe- cies and seven subspecies (Czernay 1987). However, it is suggested that morphology is not an useful tool for discrimination among species, because there is a high degree of homoplasy and morphological convergence (Grooves and Grubb 1987; Duarte et al. 2008; Merino and Rossi 2010; Gonzalez et al. 2016). Several cytogenetically studied species have shown an extensive intraspecific poly- morphism (Taylor et al. 1969; Jorge and Benirschke 1977; Neitzel 1987; Duarte and Jorge 1996; Duarte and Merino 1997; Abril and Duarte 2008; Abril et al. 2010; Valeri et al. 2018). This polymorphism would suggest a rapid speciation process driv- en by chromosomal rearrangements, with a diploid number of chromosomes rang- ing between 42 and 52, considered within six geographically established cytotypes (Duarte et al. 2008; Abril et al. 2010). As a result of this chromosomal variation, reproductive isolation has occurred in animals due to sterility problems caused by chromosomal meiosis pairing between different chromosomal lineages (Aquino et al. 2013; Cursino et al. 2014; Salviano et al. 2017). The populations in south-central South America are reproductively isolated from the Amazonian populations, being different species (Cursino et al. 2014; Salviano et al. 2017), that until now have not been described or nominated. These results have been corroborated by mitochondrial DNA variation, which clearly demonstrates the differences between some populations of M. americana Erx- leben, 1777 (Duarte et al. 2008, Abril et al. 2010), underlying the importance of research to characterize these taxa. Previous cytogenetic research has identified vari- ous M. americana cytotypes as potentially valid unamed species, and it is necessary to evaluate karyotypically previously designated names for Mazama. Therefore, animals should be sampled in all location types for each of the names currently positioned in the synonym of M. americana (Varela et al. 2010). These taxonomic uncertainties resulted in the species being categorized by the In- ternational Union for Conservation of Nature as “deficient data’, since the identity of the current taxon had no value as an evolutionary unit (Duarte and Vogliotti 2016). 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 col- lect any specimens; however, he used a series of reports by other authors as a basis for Red brocket deer neotype 145 their description, mentioning the following characteristics: “MM. 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 col- lique fupra fufci, colli fubtus albi, corporis crurumque rufofufci, vngulae nigrae, timidif- fimus, celerrimus, agilis, natat per fluuios, caro bona”. Cayenne, in French Guiana, was considered by Erxleben as the type locality for MM. americana. The absence of the species holotype for a more complete morphological and genet- ic 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 (Duarte et al. 2008; Abril et al 2010). The comparisons demonstrate that none of them belong to the same taxon described by the type location M. americana, a revision of the taxonomy of this red brocket complex being required. Material and methods Obtaining the animal and samples An adult male specimen of M. americana (Fig. 1) was collected in the city of Régina, French Guiana, 70 km from Cayenne (type locality) on 14/02/2015 by a local hunter. In French Guiana, this species is not protected by law, and can be collected without permit, the requirement being to capture outside protected areas. After collection of the individual, skin biopsies were collected and frozen in liquid nitrogen (Duarte et al. 1999) Muscle and liver samples were taken, as well as the specimen’s biometric data. The material analyzed in this study is deposited in the Museum of the Deer Research and Conservation Center (NUPECCE) — at Sao Paulo State University (UNESP), Jaboticabal campus, Brazil, recorded under catalog number NPC079. In accordance with the “Loi pour la reconquéte de la Biodiversité” (2017) and, in compliance with Access and Benefits Sharing (above-mentioned law, titre V, article 37), a tissue sample is also kept in the collection JAGUARS, belonging to Kwata NGO, Cayenne, French Guiana, under the reference M3426_JAG. Biometry 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 quan- titative 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 Sta- tistics, PAST 3.20 program (Hammer et al. 2001). 146 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) Figure |. Lateral view of the adult male Mazama americana collected in French Guiana and proposed as neotype. Analysis of the external morphology of the specimen was performed based on cri- teria used by Rossi (2000), using the photos taken immediately after collection and the entire taxidermized skin. In addition, the chromogenetic fields of the head and body were analyzed according to the nomenclature used by Hershkovitz (1982). Cranial morphology Thirty-six cranial measurements were recorded using a digital caliper (0.01 mm ac- curacy), based on the criteria proposed by Von Den Driesch (1976). A cluster analysis using the PAST program (Hammer et al. 2001) was performed based on quantitative cranial measurements of the specimen collected and of 15 animals (male and female adults) belonging to various M.. americana cytotypes: one M. bororo Duarte, 1996, two M. gouazoubira Fisher, 1814 and one M. nemorivaga Cuvier, 1817, which are stored in the NUPECCE museum (UNESP /Jaboticabal). Cytogenetic analysis Metaphase chromosome slides were prepared from tissue culture (Duarte and Jorge 2003), generated through biopsy according to Verma and Babu (1995). Chromosomal preparations were subjected to conventional Giemsa staining, G-banding using trypsin digestion (Seabright 1971), C-banding by barium hydroxide solution (Sumner 1972) Red brocket deer neotype 147 and Ag-RON silver nitrate staining (Howell and Black 1980). The chromosomes were classified as metacentric, submetacentric or acrocentric according to their arm relation- ships (Levan et al. 1964) and organized into groups according to their relative lengths (CR): Group A (large two-armed chromosomes with CR > 6%); Group C (small two- armed chromosomes with CR < 6%); Group D (large acrocentric chromosomes with CR > 5%); Group E (small acrocentric chromosomes with CR < 5%); and Group B (microcromosomes or extranumerary chromosomes with CR < 1.5%). B chromo- somes were not considered in the diploid and fundamental number calculation due to the variability between metaphases of the same individual (Abril et al. 2010). Karyo- types were carried out based on the G-bands, which were used to make the schematic representation of the G-band patterns of the neotype. The cytogenetic data of the neo- type were compared with the cytogenetic patterns of the MZ. americana cytotypes. We proposed the chromosomal evolution of the neotype from the hypothetical ancestor suggested by Abril et al. (2010). DNA extraction, amplification and sequencing DNA was extracted from a muscle sample following the protocol of Sambrook et al. (1989). The sample was subjected to the PCR technique (Mullis et al. 1986), where two mitochondrial DNA fragments were amplified: cytochrome b (Cyt-6,1140 pb; Kocher et al. 1989; Duarte et al. 2008) and a control region (D-Loop, 690 bp; Vila et al. 1999). DNA amplification used a final volume of 20 ul, containing 12 pl of H,O, 0.5 mM of dNTP, LX of reaction buffer, 1.5 mM of MgCl, 0.25 mM of each primer, 0.1 U of Taq polymerase and 3ul of DNA (15 ng/ul). The PCR protocol was 5 min at 94 °C, 35 cycles at 94 °C for 1 min, 54 °C and a final extension of 10 min at 72 °C. PCR prod- ucts were submitted to 2% agarose gel electrophoresis for amplicon identification. The purification of the amplified samples followed the Dorado—Pérez (2012) protocol. Puri- fied samples were sequenced on an automated Applied Biosystems 3730XL sequencer. Molecular data analysis 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 (Higgins et al. 1992) included in Bioedit (Hall 1999). The sequences obtained were organized into a matrix along with all sequences of the VM. americana species currently published in the GenBank System world databases (Suppl. material 1: Table $1). The best molecular evolution model was selected for the data set of each gene fragment using the jModel Test v. 0.1.1 (Po- sada and Crandall 1998), following the corrected Akaike information criterion, AICc (Akaike 1973). Sequences for the mitochondrial Cyt-b gene were obtained by con- catenating data from the two fragments using internal primers L14724 and H15149 for the 3’ end and FAR-L and FAR-H for the 5’end, obtaining a 1140-bp fragment. 148 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) The phylogenetic relationships between the different /. americana populations and the neotype were studied by Bayesian inference analysis (Huelsenbeck and Ron- quist 2001), using the program MrBayes on XSEDE 3.2.6 (Ronquist and Huelsenbeck 2003), through the online program CIPRES Science Gateway (Miller et al. 2010). 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 Fig ree v. 1.4.0 program (Rambaut 2009). Rangifer tarandus Linnaeus, 1758, Ozotoc- eros bezoarticus Linnaeus, 1758 and Mazama gouazoubira were used as an external group. Results Morphology The collected animal is presented in Figure 1 and the skull in Figure 2. The biometric and cranial measures are shown in the Suppl. material 2: Tables S2, Suppl. material 3: Tables S3. 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 poste- rior 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, short- er 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. Some- what 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 — Sao 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 (Cyi-6), MN726914 (D-Loop) Red brocket deer neotype 149 Figure 2. Right lateral (A), left lateral (B), ventral (C) and dorsal (D) views of the skull of the M@. ameri- cana neotype. Scale illustrates measurement in centimeters. Karyotype: 2n = 45 + 3Bs, FN = 51, sexual system XY_Y, 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 con- sidered 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 MM. americana. The distance tree (Fig. 3) shows substantial overlap between different geographic groups. Specimens from different localities in Brazil are widely scattered across tree branches, despite known to be cytogenetically different. Some specimens of M. ameri- cana are superimposed on the M. bororo sample, showing the morphological proximity between M. americana and this already well-established taxon (Duarte and Jorge 2003; Vogliotti and Duarte 2009). The morphological distance tree based on cranial meas- urements of the individuals shows the same result, with non-differentiation between the red brocket deer specimens and cytotypes (Fig. 4). Cytogenetics The collected animal presented a cytogenetic constitution with diploid number (2n) of 45 chromosomes and 51 chromosomal arms (fundamental number, FN). The bio- 150 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) Distance 7329 M1. americana Rio Branco- AC - Brazil oy Saat T270 MM. americana Foz de |guacu—PR -Brazil — T206 M. americana Ariquemes — RO - Brazil T0435 Mf. amerconoa Carajas—PA- Brazil 7328 M. americana Rio Branco— AC - Brazil TO/0O A. omencono Paraguay LH 7269 M. americano Buritis—RO- Brazil -— 1034 MM. americana Manaus— AM - Brazil TO31 M. améncana Rio Negro -.AM - Brazil —— T7343 M. americana Porto Velho — RO - Brazil TO21 MM. amencona foologico Ariquemes — RO - Brazil T1793 M. americana Paraguay Ti6? M. omericono Maraba —PA - Brazil 7268 MM. amencono Parque Nacional lguacu=—PA- Brazil! —— TO40 M. americana Carajas—PA - Brazil _______ 7030 MM. americana Oriximina—PA - Brazil —_—— T2711 M. americana Ariquemes — RO - Brazil 7228 ML aomencono Carajas—PA - Brazil T2277 M. americana Carajas—PA - Brazil T2760 MM. americana Itaituba—PA - Brazil 7274 M. amencana Acailandia—MaA - Brazil TO33 M. americana Manaus—AM - Brazil T7039 MM. omericona Parauapebas—PA - Brazil T255 MM. americana Anapolis—GO - Brazil T0293 M. americana Rio Branco— AC- Brazil 7254 Mf. americongd Imperatriz— MA - Brazil TO44 M. americana Parquede Carajas—PA - Brazil T327 Af. omencaono Rio Purus— Figure 8. Phylogenetic tree from Bayesian inference of the fragments of the concatenated D-Loop and Cyt-6 regions. The values represent the posterior probability of the analysis. Discussion Neotype designation Mazama americana Erxleben, 1777 is the name given to a species commonly recog- nized 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 (Duarte et al. 2008; Abril et al. 2010), due to the morphological convergence within the genus as well as the recent divergence through chromosomal changes (Gonzalez and Duarte, in press). The holotype of the species is unknown, as is any specimen of the type series, precluding any current comparison. The taxonomic complexity of the species, indicative of the existence of a complex of several cryptic species, is a prima facie case and can only be solved by designating a neotype (Interna- tional Code of Zoological Nomenclature 1999, art. 75.3.1). Erxleben’s (1777) original description of M. americana is very brief and does not in- clude the many characteristics currently used to diagnose species of the genus Mazama, as he did not see any specimens but based his description on a series of reports by other authors, such as Des Marchais, 1725, who described the “Biche de Guinee’, as well as on a drawing of a juvenile made by Seba in 1730 called “Cervula surinamensis”, which simply illustrates a red deer. Linnaeus is known to have acquired specimens of Seba, but he never mentioned Cervula surinamensis, whose whereabouts were unknown, so it is suggested that there was no specimen that served as the basis for Erxleben’s description. Red brocket deer neotype 155 Thus, the absence of any specimen of the type series, the lack of critical informa- tion in the original description, as well as the large taxonomic uncertainties in this species complex (Abril et al. 2010; Cursino et al. 2014; Salviano et al. 2017), indicate that their identity can only be clarified by designating a neotype. Morphology The genus Mazama is characterized by rapid diversification and morphological paral- lelism (Duarte et al. 2008; Gutiérrez et al. 2017), thus generating species complexes, such as in the case of M. americana, which present major challenges for today’s science (Abril et al. 2010; Cursino et al. 2014). The individuals belonging to the different cytotypes of MM. americana found in Brazil (Abril et al. 2010) and the neotype analyzed herein could not be separated in the analyses made by morphological characteristics, as already observed by Rossi (2000) and Duarte et al. (2008), suggesting great morphological similarity between the differ- ent species of the /. americana complex. 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 indi- vidual cranial morphometric characteristics, which do not appear to have any geographi- cal relation. Similarly, Rossi (2000) reported the difficulty in detecting any pattern of sim- ilarity between M. americana samples according to cranial morphological characteristics. 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 (Duarte et al. 2008). Cytogenetics The chromosomal polymorphism found in red brocket deer is surprising and shows high levels of intra- and interspecific chromosome variation (Duarte et al. 2008; Abril et al. 2010). Thus, the neotype according to its karyotypic pattern does not fit into any of the red brocket deer cytotypes studied in Brazil (Abril et al. 2010), thus recognizing that these variants should be considered as different species according to cytogenetic evidence, since differences of more than two chromosomal pairs generate an efficient postzygotic reproductive barrier (Cursino et al. 2014; Salviano et al. 2017). 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 Neitzel (1987), in chromosomes formed from tandem fusions the interstitial C bands shrink in size over time until they are so weak that they may disappear completely. As previously reported by Sarria-Perea (2004), cytogenetic analysis of M. americana cytotypes reveal an intense process of chromosomal evolution. The chromosomal reor- 156 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) ganization is mainly due to fusions between Group D and E chromosomes to form new chromosomes and a consequent decrease in diploid number (Abril et al. 2010). Accord- ing to their diploid number, it is possible to observe the approximation of the neotype to the group of individuals from Rondénia (2n = 42/43; NF = 46) and Juina (2n = 44/45; NF = 48), which have lower numbers of chromosomes. However, the chromo- somal changes that occurred in the formation of Rondénia and Juina cytotypes are not the same as those involved in the karyotype formation of the Z. americana neotype (Fig. 9), clearly showing the isolation of populations, which is also geographically con- firmed, with the Amazon River in their midst acting as the major geographical barrier. Through the use of chromosomal banding techniques, important results were gen- erated for the karyotype study of M. americana. Based on their comparison, it was ob- served that there is a chromosomal difference between the six . americana cytotypes and the neotype due to pericentric inversions, tandem fusions and centric fusions, as previously reported by Sarria-Perea (2004) and Abril et al. (2010). Based on the hypothetical ancestor of M. americana and the chromosomal evolution among the cy- totypes proposed by Abril et al. (2010), the rearrangements involved in the karyotype formation of the neotype were two pericentric inversions, two tandem fusions and two centric fusions, thus showing greater evolutionary proximity of the neotype to the /. americana strain, with a higher number of chromosomes (Parana, Santarém, Jari and Carajas; Fig. 9). The large chromosomal variation found in M. americana can be explained by the theory of chromosomal fragility proposed for Mazama (Duarte and Jorge 1996; Var- gas-Munar et al. 2010; Tomazella et al. 2017), which would induce the occurrence of breakages and chromosomal exchanges. These chromosomal rearrangements could have led to the formation of new species in a relatively short time after geographical isolation, given that chromosomal changes can promote incipient divergence and lead ultimately to species diversification (Potter et al. 2017). Consequently, it is clear that the proposed neotype does not belong to the same species as the known M. americana cytotypes (Duarte et al. 2008; Abril et al. 2010), since the number of chromosomal pairs involved in these differences certainly generate an insurmountable postzygotic reproductive barrier due to sterility of the hybrid, as evidenced by Salviano et al. (2011) and Cursino et al. (2014) in the same specific com- plex. Thus, cytogenetics is the more important characteristic for the reclassification of the individuals of the group at the species level. With MZ. americana of Cayenne as the first description (Erxleben 1777), it is now necessary to redescribe and name all other species that are different from this neotype. Molecular phylogeny 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 MZ. americana, yielding results very similar to previous studies (Carnelossi 2008; Duarte et al. 2008; Abril et al. 2010), but comple- Red brocket deer neotype 157 clin Ie been tn at HL thvvetta sevens CEE CA ORDO DE OE KPa Oe WMH ioa0 of dl ia as oe oe of #8 Hh ita oo She 08 . 2 ; Rondonia {LS tate] | ut MN ni Th) fut " Ti | oie oh ok oe oe i | t a mn 6 de LD GVEC pean neee| i nee tH] at a oe sh aa aa 00 is of t Hypothetical Ancestor M. americana. bi oo 08 fo Ot oe a x 7 i na & Tl ‘ wy. : ve At. 0 04 08 66 66 48 F864 28 oe 2n=52/53 NF=54 Parana ee ae *s os acne 2 ee ees eee es | * Carajas " i t ab ie an os eam Tandem Fusion “We On ean a0 ts as oe as am Centric Fusion © Pericentric Inversion Neotype Figure 9. Chromosomal evolution showing the relationships of the six cytotypes compared to the americana neotype. Juine: 2n = 44/45; Rondénia: 2n = 42/43; Santarém: 2n = 50/51; Jari: 2n = 48/49; Parana: 2n = 52/53; Carajas: 2n = 50/51; Neotype: 2n = 45 (Adapted from Abril et al. 2010). mented now by information of the neotype. However, these analyses make clear the nu- merous gaps in taxonomic and evolutionary knowledge of the M. americana complex. The specimen of M. americana analyzed in this study complies with all the condi- tions 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 aM. americana pattern (neotype), it is possible to define where the current cytotypes will be positioned. It 158 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) 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 tax- onomy (Gutiérrez et al. 2017) It should be reiterated that it remains a formidable challenge to resolve the relation- ships 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 Duarte et al. (2008), Carnelossi (2008), Abril et al. (2010), Cursino et al. (2014) and Salviano et al. (2017), since until now the neotype does not match with any known cytotype. 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. Acknowledgments We thank Coaraci who collected the animal for this study. Fundacéo de amparo 4 Pes- quisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico (CNPq) provided financial support. We also thank Joao Airton Boer for his support for the laboratory work at the NUPECCE/UNESP. We thank Na- talia Aranha de Azevedo for the drawings of the animal heads used in the molecular tree. References Abril VV, Carnelossi EAG, Gonzalez $, Duarte JMB (2010) Elucidating the evolution of the red brocket deer Mazama americana complex (Artiodactyla; Cervidae). 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Mastozoologia Neotropical 16: 499-503. Von Den Driesch A (1976) A guide to the measurement of animal bones from archeological sites, as developed by the Institut fur Palaeoanatomie, Domestikations for schung und Ge- schichte der Tiermediz in of the University of Munich. Peabody Museum Bulletin 1: 35-37. Supplementary material | Table $1. Samples used in this study for genetic analysis Authors: Analorena Cifuentes-Rincén, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Mauricio Barbanti Duarte Data type: molecular data Explanation note: Nupecce’s identification, species, gene accessed, accession number in genbank, origin (locality) and source (when collected). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.958.50300.suppl1 Supplementary material 2 Table S2. Biometric data of the M. americana neotype Authors: Analorena Cifuentes-Rincén, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Mauricio Barbanti Duarte Data type: species data Explanation note: Measured in cm and mass in kg. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.958.50300.suppl2 Red brocket deer neotype 163 Supplementary material 3 Table $3. Cranial measurements of M. americana neotype represented in millimeters Authors: Analorena Cifuentes-Rincén, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Mauricio Barbanti Duarte Data type: species data Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.958.50300.suppl3 Supplementary material 4 Figure S1. Phylogenetic tree of the Cyt-b gene Authors: Analorena Cifuentes-Rincén, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Mauricio Barbanti Duarte 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. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.958.50300.suppl4 164 Analorena Cifuentes-Rincén et al. / ZooKeys 958: 143-164 (2020) Supplementary material 5 Figure S2. Phylogenetic tree of the D-/oop gene Authors: Analorena Cifuentes-Rincén, Jorge Alfonso Morales-Donoso, Eluzai Dinai Pinto Sandoval, Iara Maluf Tomazella, Aline Meira Bonfim Mantellatto, Benoit de Thoisy, José Mauricio Barbanti Duarte 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. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.958.50300.suppl5