Fecal DNA reveals wider distribution and a new Evolutionary Significant Unit for an endemic deer of the Atlantic Forest (Mazama jucunda)

Fecal DNA reveals wider distribution and a new Evolutionary Significant Unit for an endemic deer of the Atlantic Forest (Mazama jucunda) | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Animal Genetics This is a preprint and has not been peer reviewed. Data may be preliminary. 5 May 2025 V1 Latest version Share on Fecal DNA reveals wider distribution and a new Evolutionary Significant Unit for an endemic deer of the Atlantic Forest (Mazama jucunda) Authors : Jeferson L. S. Freitas 0000-0002-7491-3553 , Pedro H. F. Peres , Francisco Grotta-Neto , Márcio L. Oliveira , and Jose Mauricio Barbanti Duarte [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174645748.86229982/v1 369 views 245 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Limited knowledge of the geographical distribution of brocket deer persists due to their elusive behavior and the morphological similarity among species. A population of red brocket deer from the genus Mazama was recently found in a protected area of the Brazilian Atlantic Forest, the Rio Doce State Park (Brazilian acronym PERD), but identification to species level was not available. Since the Atlantic Forest harbors two endemic and threatened red brocket deer, this study aimed to identify which species occur in PERD through fecal DNA analysis. Fecal samples collected in PERD and throughout the Atlantic Forest were sequenced for six mitochondrial DNA region fragments (1450bp) and analyzed towards a group of reference specimens. We performed phylogenetic analyses and implemented coalescent methods of molecular species delimitation (GMYC and bPTP) to identify molecular operational taxonomic units (MOTUs). We also constructed a haplotype network and a genetic distance matrix. The phylogenetic hypothesis recovered the samples from PERD linked to the Small Red Brocket deer ( Mazama jucunda ), but in a reciprocally monophyletic topology, corresponding to two exclusive MOTUs. PERD has unique haplotypes and its genetic distance to M. jucunda is similar to that between M. jucunda and its most closely related species, the Brazilian Dwarf Brocket ( Mazama nana ). Herein we report an evolutionary significant unit (ESU) of M. jucunda 700 km apart from the species current distribution. The observed genetic structure and the isolation context of PERD raise the hypothesis of a new red brocket species that should be further tested, including cytogenetic data. This study represents an important advance in the knowledge of the geographical distribution and genetic structure of a threatened Neotropical forest deer endemic to a devasted hotspot with direct and short-term implications for its conservation. Introduction The Atlantic Forest is one of the most biodiverse and threatened tropical forests in the world. The high endemism indices (Mittermeier et al. , 2011) and the severe loss of approximately 88% of its original coverage (Ribeiro et al. , 2009) make it one of the most important hotspots for biodiversity conservation (Myers et al. , 2000). Ungulates are key species in neotropical forests due to their various ecological roles, such as soil displacement, herbivory, seed dispersal, and as prey for large predators (Peres, 2000; Beck, 2006; Beck, Thebpanya, & Filiaggi, 2010; Beck, Snodgrass, & Thebpanya, 2013). Among them, brocket deer have evolved under strong selective pressures to adapt to dense forest environments (Oliveira et al. , 2025) and are considered indicators of forest preservation due to their ecological demands for pristine areas (Duarte et al. , 2017; Oliveira, Couto, & Duarte, 2019). In the Atlantic Forest, three red brocket deer species (genus Mazama ) are found: M. rufa , M. nana , and M. jucunda (Duarte et al., 2017; Oliveira et al., 2019; Peres et al., 2021). Both M. nana and M. jucunda are endemic to this biome and are currently classified as Vulnerable by the International Union for Conservation of Nature and Natural Resources (IUCN) (Duarte et al. , 2015; Vogliotti, Oliveira, & Duarte, 2016). Moreover, M. jucunda is not only a threatened and endemic species (the largest endemic mammal in the Atlantic Forest) but it contains one of the most limited distributions among deer species in the world (Weber & Gonzalez, 2003). Despite their ecological significance, these species face severe threats, including poaching, habitat loss, disease transmission from domestic livestock, and predation by domestic dogs (Duarte et al. , 2015; Duarte & Vogliotti, 2015; Vogliotti, Oliveira, & Duarte, 2016). In addition to these threats, the conservation of Mazama species is further hindered by the historical presence of significant taxonomic gaps (González & Duarte, 2020). These gaps are mostly related to the fact that these animals are extremely elusive and still have a high degree of homoplasy in morphological characters, while being very different molecular and cytogenetically (Duarte et al., 2008; Peres et al 2021; Bernegossi et al 2024). For example, cryptic species complexes are identified, that is, two or more distinct species that are misclassified (and hidden) under the same species name (Bickford et al. , 2007). This is the case of the species M. americana (Duarte, González, & Maldonado, 2008; Gutiérrez et al. , 2017), for which different chromosomal patterns have been identified, called cytotypes ( M. americana sensu lato ), with geographical coherence throughout the distribution of the species in Brazil (Bernegossi et al. , 2024). Recently, Cifuentes-Rincón et al. (2020) indicated a neotype for the species, thus fixing its the chromosomal pattern ( M. americana sensu stricto ). In addition to the taxonomic issues, information on the geographical distribution of these species is still scarce; only M. jucunda and M. nana had their areas of occurrence modeled in studies carried out specifically with this purpose (Duarte et al. , 2017; Oliveira, Couto, & Duarte, 2019). The forest habitats, often dense and difficult to access, and the elusive behavior of these species make it very costly to capture individuals to obtain morphological data and live cells to perform cytogenetic analyzes, which are very informative in the taxonomic resolution of the group (Cifuentes-Rincón et al. , 2020; Peres et al. , 2021 b ; Sandoval et al. , 2022; Bernegossi et al. , 2023; Morales-Donoso et al. , 2023). In this regard, non-invasive sampling methods stand out. Camera traps can assist in the identification of recorded species and provide ecological data (Grotta‐Neto et al. , 2020). The identification of forest deer by this method, however, is limited due to the extreme similarity, and very similar coloring pattern, such as the case with the species M. americana , M. rufa and M. jucunda (Fig. 1; Peres et al., 2021b). Another widely used methodology is fecal sampling followed by species-level genetic identification using mitochondrial DNA markers (Oliveira et al. , 2022). This methodology also allows estimates of population density and geographic distribution modeling (Duarte et al. , 2017; Oliveira, Couto, & Duarte, 2019; Silva, Oliveira, & Duarte, 2020; Morini, Grotta‐Neto, & Duarte, 2024). Fig. 1 Individuals of Rio Doce State Park recorded by camera traps (A) and species of the genus Mazama with similar size and external morphology (B = Mazama americana , C = Mazama rufa and D = Mazama jucunda ). Scale is centimeters. Source: Wild Animal Conservation Institute (ICAS; images in A) and Deer Research and Conservation Center (NUPECCE; images in B, C and D). In the context of the Atlantic Forest, an important gap regarding the geographical distribution of red brocket deer is the identification of species inhabiting the PERD, one of the largest continuous areas of this biome (Ribeiro et al. , 2009). Using fecal DNA, Oliveira et al. (2020) performed phylogenetic analysis with fragments of the mitochondrial cytochrome-b gene and demonstrated that this population belongs to the genus Mazama , but they were not able to carry out the identification at the species level because the mitochondrial marker used was not sufficiently informative, being necessary to add more polymorphic regions (Peres et al., 2021a). Moreover, even though we had access to images of the animal in the area (Fig. 1), it was not possible to perform the identification due to high morphological similarity to other close-related species (Peres et al 2021). In this background, this study aimed to identify the deer species inhabiting the PERD using fecal DNA genetic analysis and a comprehensive sampling of potential species. Materials and methods Study area The Rio Doce State Park (PERD) is the main continuous area of Atlantic Forest in the state of Minas Gerais with 35,976 ha. The predominant vegetation typology in the area is the Submontane Semi-deciduous Seasonal Forest. The fauna of PERD is very diverse, including endemic taxa to the Atlantic Forest and several species are threatened with extinction, like the Northern Muriqui ( Brachyteles hypoxanthus ), the Brown Howler Monkey ( Alouatta guariba ), the Giant Armadillo ( Priodontes maximus ) and the Jaguar ( Panthera onca ), and other ungulates like the Lowland Tapir ( Tapirus terrestris ). Although the park’s Management Plan (IEF, 2023) indicates the presence of only one deer species, the Red Brocket deer ( Mazama americana ), there are also indications of the presence of another forest deer, the Gray Brocket deer ( Subulo gouazoubira ), whose presence inside the unit needs to be confirmed. Fecal and Reference samples Fecal samples were collected using a scat detection dog specially trained to locate feces of all Brazilian deer species, conditioned to not interact with wildlife, and subjected to strict sanitary control. The searches took place along 20 transects of up to 1 km throughout the entire park. The dog searched for samples freely, without a leash, moving within a maximum radius of 20 meters around the handler. Each sample was georeferenced, identified with the acronym PERDJ, collected without direct hand contact, and stored in 50 mL plastic tubes containing absolute ethanol. Samples previously collected and identified to species level, deposited in the Fecal Bank of the Deer Research and Conservation Center (NUPECCE) of the São Paulo State University (UNESP), were also used in this study. Some of these samples are from the PERD (Oliveira et al., 2020) and the remaining of them are from other protected areas in the states of São Paulo, Paraná and Santa Catarina (Oliveira, 2015; Grotta‐Neto et al. , 2024). We included a reference dataset composed of Mazama specimens with reliable identification provided by morphological and cytogenetic evaluation. All specimens were from the database of samples and sequences managed by the Deer Research and Conservation Center (NUPECCE) of the São Paulo State University (UNESP). We selected a total of 36 samples, including two M. americana sensu stricto, five M. jucunda , eight M. nana , four M. rufa , and three M. temama . We also included individuals from M. americana sensu lato cytotypes: Acre (three samples), Carajás (four), Juína (three), and Rondônia (four). DNA extraction, amplification and sequencing All laboratory procedures regarding fecal samples were conducted in a dedicated room for fecal DNA analysis and had contamination controls. Fecal DNA extraction was performed with a non-commercial silica-based protocol adapted from Boom et al. (1990) and Höss and Pääbo (1993). DNA was already available at the NUPECCE bank from previous tissue extractions for the reference samples that required sequencing. First, all fecal samples were screened to exclude samples of Subulo gouazoubira using a PCR-RFLP protocol based on a 224 bp fragment of the mitochondrial cytochrome b gene (González et al. , 2009). For the full amplification and sequencing of M. jucunda samples, we selected five spatially distributed samples from PERD and additional six fecal samples of M. jucunda from other protected areas throughout the species’ range in the Atlantic Forest. Both fecal and reference sample DNA amplification was performed by PCR using primers designed to amplify fragments (149 to 463 base pairs) of the mitochondrial genes cytochrome b (CYTB), NADH dehydrogenase subunit 5 and 2 (ND5 and ND2) and cytochrome c oxidase subunit 1 (COI) (Table S1). The PCR reactions were standardized to a final volume of 30 µL (25 µL of mix and 5 µL of DNA), containing 1x buffer and 1.5 U of Taq DNA polymerase (both Platinum TM , Thermo Fisher Scientific); 2 mM MgCl 2 ; 0.6 mM dNTP; 1.3 mg/ml BSA and 0.5 pM of each primer. The reactions were performed in a Bio-Rad C1000 Touch™ Thermal Cycler in a touch-down protocol, which totaled 45 cycles under the following conditions: an initial step of 94 °C for 2 min; followed by 5 cycles of 94°C-50 s/ 58°C-50 s/ 72°C-50 s; 6 cycles of 94°C-50 s/ 57°C-45 s/ 72°C-50 s; 8 cycles of 94°C-50 s/ 56°C-40 s/ 72°C-50 s; 13 cycles of 94°C-50 s/ 55°C-35 s/ 72°C-50 s; 13 cycles of 94°C-50 s/ 54°C-30 s/ 72°C-50 s and finally a final extension at 72 °C for 10 min. Finally, the PCR products were visually confirmed by electrophoresis on 2% agarose gel, purified using the kit Wizard ® SV Gel and PCR Clean-Up System (Promega), and sequenced (forward and reverse) by the Sanger method in a sequencer ABI 3500 Genetic Analyzer (Applied Biosystems™). The resulting electropherograms were visually reviewed, the primers were removed, and the consensus sequences were generated in BioEdit 7.2.5 (Hall, 1999). Except for three reference animals that required Sanger sequencing for two gene fragments, we extracted mtDNA regions of interest from mitogenomes deposited in the NUPECCE bank. Details on the mitogenome assembly are described in Bernegossi et al. (2023). All produced and extracted sequences were deposited in GenBank (Table S2 and S3). Characterization of the DNA matrix We added two extra reference samples from GenBank, one M. americana sensu stricto and one M. temama and three other species of the family Cervidae to compose the outgroup in phylogenetic analysis: Alces alces, Capreolus pygargus and Rangifer tarandus . The final dataset was composed of 41 reference samples and 11 fecal samples, five of which were from PERD (Tables S2 and S3). The location of samples used in this study that were collected at the PERD and other samples identified as M. jucunda are shown in Fig. 2. Fig. 2. Samples from Rio Doce State Park (PERD) and samples of Mazama jucunda used in this Study. The consensus sequence of each sample, the sequences recovered from mitogenomes, and the sequences obtained in GenBank were grouped by each gene fragment and aligned in MAFT online server (Katoh et al., 2019; https://mafft.cbrc.jp/alignment/server/). The alignments were reviewed and edited in BioEdit and then concatenated in the Mesquite 2.75 (Maddison & Maddison, 2023), thus forming the final matrix with 1450 bp. The matrix polymorphism was analyzed in the MEGA 11 (Tamura, Stecher, & Kumar, 2021) by counting the variable sites and parsimony-informative sites (PICs; sites with mutations in at least two samples). Finally, the evolutionary model was estimated in the program MrModelTest 2.4 (Nylander, 2004), being selected the one with the lowest AIC value among the 24 models tested. Phylogenetic analysis The phylogenetic analyses were performed by Bayesian Inference (BI) in the BEAST 1.10.4 package (Suchard et al. , 2018). The run parameters were set in program BEAUti (BEAST package), and the analyses were implemented in three different runs on the CIPRES Science Gateway online server (Miller, Pfeiffer, & Schwartz, 2010). Each Monte Carlo Markov Chains (MCMC) run had 50 million generations, with sampling at every thousand generations. We implemented the lognormal uncorrelated relaxed clock and the Yule process speciation tree model. The results of the three independent runs were combined in the Log Combiner program (BEAST package) by applying a burn-in of 25% and the convergence of the analysis was verified in the program Tracer 1.7.2 (Rambaut et al. , 2018), being considered satisfactory when it presented values from 200 in the ESS parameter ( Estimated Sample Size ). The statistical support values of the clades – posterior probability (PP) – were annotated in the Tree Annotator program (BEAST package) and the final tree was visualized and graphically edited in FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). In addition to the ultrametric phylogenetic tree generated in BEAST, a non-ultrametric tree was also generated in the program MrBayes (Ronquist et al. , 2012) to be used as input in one of the methods of molecular species delimitation. The analysis was also performed on the CIPRES Science Gateway online server, and was composed of two independent runs with four MCMC chains and 30 million generations sampled every thousand generations and combined after a 25% burn-in. Convergence was verified when the final average standard deviation of split frequencies parameter reached a value bellow 0,01. Molecular species delimitation Molecular species delimitation analyses for the identification of MOTUs (molecular operational taxonomic units) was performed through two approaches, GMYC ( General Mixed Yule Coalescent , Fujisawa and Barraclough, 2013) and bPTP (Bayesian implementation of Poisson Tree Process ; Zhang et al., 2013). GMYC analysis was performed using the splits package (Ezard, Fujisawa, & Barraclough, 2021) in R 4.2.2 (R Core Team, 2024). We used an ultrametric phylogenetic tree generated by BI in BEAST, without the outgroup, and two delimitation thresholds were tested (single and multiple). The probabilities of the GMYC models were compared against the probability of a null model, in which only one MOTU is identified, and the hypothesis that the models were statistically different was accepted when they presented p < 0.05. The bPTP method was performed in its own online server (https://species.h-its.org/). We tested the use of an ultrametric phylogenetic tree generated by BI in BEAST and a non-ultrametric tree generated by BI in MrBayes, which had the outgroup removed at the time of implementation of the analysis. Thus, in summary, four species delimitation hypotheses were generated: (1) single threshold and (2) multiple thresholds of the GMYC method and (3) non-ultrametric tree and (4) ultrametric tree by the bPTP approach. Haplotype network and genetic distance For both analyses, we used the final matrix without the outgroup, and the sequences were grouped by the assigned species (reference samples and previously identified fecal samples) or as coming from PERD. The haplotype network was generated in PopART 1.7 (Leigh and Bryant, 2015) by the method median-joining (Bandelt, Forster, & Rohl, 1999). Each grouping was assigned a color, chosen automatically in the program, to facilitate the interpretation of the network. The mean pairwise genetic distance between clusters was estimated using the Kimura 2-parameter (K2P) model in the MEGA 11. Fecal samples collection and screening The fecal sampling survey encompassed approximately 24 km, over 40 hours of search effort, yielding a total of 108 fecal piles collected (4.5 samples/kilometer and 2.7 samples/hour). Five samples were identified as S. gouazoubira (PERDJ51, 54, 55, 56 and 58) and 3 samples failed the PCR amplification step (PERDJ49, 62 and 75), resulting in 100 samples, presumably M. jucunda , that were available for next step – sequencing and genetic analysis (Table S4). A previous study of our research group (Oliveira et al. , 2020) collected 12 samples in PERD, of which five (PERD2, 4, 6, 7 and 11) were identified as belonging to the genus Mazama through phylogenetic analysis using two fragments of the mitochondrial cytochrome c (CYTB) gene. Therefore, considering the available samples, their spatial distribution in the PERD and the available sequences produced, five samples from PERD were selected to compose the final matrix: PERD2, 4 and 7 from Oliveira et at. (2020), and PERDJ 12 and 17 collected in this study (Fig. 2). It is worth mentioning that the PCR-RFLP complete protocol would allow, in theory, the identification of M. jucunda samples (González et al. , 2009). However, when analyzing the sequences of PERD samples, produced by Oliveira et al. (2020) and in this work, we verified a mutation in the restriction site of the enzyme that would discriminate M. jucunda from M. nana ­ – BstN -, making it impossible the identification at species level (data not presented). Fortunately, the distribution of M. nana is restricted to the south regions of Brazil and this situation had no effect on PERD sample identification. Sequencing and DNA matrix characterization All selected fecal and reference samples were successfully sequenced. The only exception was the fragment of the COI gene for the fecal sample PERDJ17. For this sample and sequence, and only in this case, the corresponding region in the matrix was filled with the general ambiguity symbol “N”. The final matrix consisted of 52 samples (including the three outgroup samples) and was 1450 bp in total, of which 395 are variable sites and 258 are parsimony-informative sites (PICs) (Table 1). The evolutionary model selected for the final matrix was GTR+G. Table 1 Size and polymorphism of the final matrix and the five fragments that compose it. PICs = parsimony-informative sites; bp = base pairs. CYTB-224 185 bp (12.76%) 50 (12.66%) 34 (13.18%) CYTB-306 267 bp (18.41%) 89 (22.53%) 55 (21.32%) ND5a 263 bp (18.14%) 72 (18.23%) 47 (18.22%) ND5b 207 bp (14.28%) 60 (15.19%) 44 (17.05%) ND2 423 bp (29.17%) 97 (24.56%) 65 (25.19%) COI 105 bp (7.24%) 27 (6.84%) 13 (5.04%) Final matrix 1450 bp (100%) 395 (100%) 258 (100%) *After trimming the primers. Phylogeny and molecular species delimitation The phylogenetic inference generated by BI successfully positioned the PERD population in the genus Mazama , and as the sister group of the reference M. jucunda, both with high support (PP > 0.9) (Fig. 3, BI). The analysis recovered three main clades in Mazama topology. The first (clade I) gathers samples of M. temama; the second (clade II) gathers the group with high diploid number of chromosomes (2n = 49-53): M. americana , M. rufa and M. americana Carajás cytotype; and the third (clade III) gathers the lineages with low diploid number (2n = 42-45): M. americana from the Acre, Juína and Rondônia cytotypes, M. nana , M. jucunda and the samples from PERD. The composition of clade VII stands out, which brings together, in a reciprocally monophyletic way, the samples of PERD (clade VIII) and M. jucunda (clade IX). Fig. 3. Phylogenetic analysis by Bayesian Inference (BI; generated in BEAST, ultrametric tree) and molecular species delimitation by the method General Mixed Yule Coalescent (GMYC; single threshold, p = 0.001). The statistical support values of the clades are represented by posterior probability (PP). Roman numerals (I – IX; white boxes with black borders) identify the highlighted clades and Arabic numerals (1 – 12; black boxes) identify the MOTUs (molecular operational taxonomic units), both cited throughout the text. Samples identified with T### (“T” + three digits) represent NUPECCE vouchers and samples identified with # # # # # # # # (eight digits) represent sequences obtained from GenBank. Illustration source: Natalia de Azevedo (Azevedo, Oliveira, & Duarte, 2021). The two approaches of molecular species delimitation generated quite different results: the number of MOTUs varied from 12 to 25. In the GMYC method, both single and multiple threshold hypothesis were statistically significant, with p = 0.001 and 0.04, respectively. The single threshold hypothesis resulted in 12 MOTUs; and the multiple thresholds, 15 MOTUs. In the bPTP approach, the analysis made with the non-ultrametric tree generated 18 MOTUs; with the ultrametric tree, 25 MOTUs. In this session, we will only present the results from the most coherent species delimitation approach in light of the current literature, which was the GMYC single threshold hypothesis (Fig. 3, GMYC). The other hypotheses showed an excessive number of MOTUs represented by single specimens and subdivided well-consolidated species such as M. nana , M. americana and M. temama . The single threshold GMYC hypothesis indicated two MOTUs for the clades representing PERD (clade VIII) and other M. jucunda (clade IX) samples. The summary of the four hypotheses generated can be found in the supplementary information (Fig. S1). Haplotype network and genetic distance The haplotype network (Fig. 4) shows that the PERD population possesses unique and exclusive haplotypes which are not shared with any other species. In addition, we observe that 5 mutational steps separate the haplotype grouping of PERD from the grouping of M. jucunda , while the separation between this species and M. nana is 3 mutational steps. Fig. 4. Haplotype network generated from the final matrix by the method median-joining . The traces on the branches represent mutational steps. The smallest genetic distance from the PERD population to any other species was with M. jucunda, 0.0158. This value is very close to the genetic distance between M. jucunda and its most closely related species, M. nana (0.0165) (Table 2). Table 2 Average pairwise genetic distance between species and the population of Rio Doce State Park (PERD). Mju = M. jucunda ; PERD = Rio Doce State Park; Mna = M. nana ; MamAC = M. americana Acre cytotype; MamJU = M. americana Juina cytotype; MamRO = M. americana Rondônia cytotype; MamCA = M. americana Carajás cytotype; Mru = M. rufa and Mam = M. americana. PERD 0.0158 Mna 0.0165 0.0162 MamAC 0.0226 0.0231 0.0158 MamJU 0.0260 0.0231 0.0221 0.0189 MamRO 0.0293 0.0292 0.0236 0.0205 0.0195 MamCA 0.0472 0.0464 0.0382 0.0428 0.0497 0.0496 Mru 0.0489 0.0477 0.0400 0.0429 0.0499 0.0503 0.0043 Mam 0.0499 0.0495 0.0411 0.0450 0.0511 0.0513 0.0160 0.0173 Mte 0.0593 0.0591 0.0558 0.0561 0.0583 0.0598 0.0616 0.0594 0.0562 Discussion Our phylogenetic results linked the red brocket deer from PERD to M. jucunda instead of any M. americana lineage. However, there is clear phylogenetic structuring with high statistical support, also demonstrating geographical coherence. PERD is located in Minas Gerais state, while all other M. jucunda samples come from southern regions, more than 700 km away, including São Paulo, Paraná, and Santa Catarina states. The analyses of this population conducted by Oliveira et al. (2020) (530 bp of the CYTB gene) were not sufficient to identify the species, only being able to assign the population as belonging to the genus Mazama in subtribe Odocoileina. In this study, by using six mitochondrial markers and an expanded sampling of both PERD samples and M. jucunda individuals from across the species distribution, we positioned the population robustly. The results of the species delimitation (Fig. 3, GMYC) corroborate the existence of genetic structuring between the PERD population and the other samples of M. jucunda . The GMYC approach with a single threshold was more robust than the others – GMYC with multiple thresholds and bPTP with ultrametric and non-ultrametric trees – because it presented greater congruence with the current taxonomic arrangement of the group (Merino & Rossi, 2010; Peres et al., 2021a) and chromosome lineages identified for red brocket deer (Cursino et al. , 2014; Salviano et al. , 2017; Galindo et al. , 2021; Bernegossi et al. , 2024). GMYC was the most conservative, avoiding an artificial pulverization of species proposed by phylogenetic analysis. In general, the other approaches implemented, especially those based on the bPTP method, showed excessive subdivisions (Fig. S1). The combined results from the haplotype network (Fig. 4) and the genetic distance matrix (Table 2) suggest a historical absence of gene flow between the PERD and other M. jucunda studied populations, reinforcing the existence of genetic structuring. Furthermore, there is evidence supporting the possibility that they are distinct species, as the differences observed here between the PERD population and M. jucunda are comparable to those between M. jucunda and M. nana . It is important to highlight that M. nana is the phylogenetically closest species to M. jucunda , being recovered as a sister species with reciprocal monophyly in this study (Fig. 3, BI) and in other studies in the literature (Duarte et al., 2008; Gutiérrez et al., 2017; Peres, et al., 2021a). They were separated from a hypothetical common ancestor in the early Pleistocene, about 1 million years ago (Duarte et al., 2008) and are widely recognized from a taxonomic point of view (Abril et al. , 2010; Vogliotti & Duarte, 2010). Their morphological patterns are clearly differentiated (Rossi, 2000), and their chromosomal patterns have already been described: M. jucunda has a diploid chromosome number (2n) = 32–34 and a fundamental number (FN) = 46 (Duarte & Jorge, 2003; Vogliotti & Duarte, 2010), while M. nana has 2n = 36–39 and FN = 58 (Abril et al., 2010; Abril & Duarte, 2008). Such chromosomal differences represent an important mechanism of reproductive isolation, falling within the biological species concept (Cursino et al. , 2014; Salviano et al. , 2017; Galindo et al. , 2021; Bernegossi et al. , 2024). Given this context, if the chromosomal differences between the PERD and other M. jucunda populations are as significant as the molecular genetic differences observed in this study – like those between M. jucunda and M. nana – we will likely be dealing with distinct species. Chromosome divergence has been claimed as decisive pos-zygotic barrier that would lead to populational isolation because of the reduced reproductive fitness of hybrids between divergent lineages (Cursino et al. , 2014; Salviano et al. , 2017). Given the importance of chromosomal divergences in assisting the taxonomic resolution of the genus Mazama (George e Benirschke, 1977; Duarte and Jorge, 2003; Peres et al., 2021a), it is urgent to collect living tissue from PERD animals for cytogenetic analysis. Despite this indication, until further analyses are conducted and have robust evidence, our position is that the PERD population belongs to the species M. jucunda , being considered an ESU due to its genetic structure and isolation context. The ESU concept, first introduced by (Ryder, 1986), can be generally defined as a population or group of populations that is considered distinct for conservation purposes due to its unique genetic makeup and evolutionary history (Ryder, 1986), which was demonstrated in our study the reciprocally monophyletic topology, the isolation pattern in the haplotype network and the genetic distance. Identifying ESUs is crucial for developing effective conservation strategies, such as captive breeding, habitat protection, and translocation efforts, as it ensures that conservation actions maintain genetic diversity and evolutionary potential (Hutama et al. , 2017; Forester et al. , 2022). Indeed, this concept has been applied to deer species (Zachos et al. , 2014; Singh et al. , 2021), including Neotropical ones (Oliveira et al. , 2020; Gonzalez et al. , 2024). The exclusive use of mitochondrial markers for genetic analysis and the absence of cytogenetic data, which are very informative for taxonomic definitions in this group, are important limitations of this study. Thus, the interpretation of the results should be done with due caution, since gene trees based on mitochondrial regions may overestimate structuring when there is female philopatry (Hoelzer, 1997; Eberle et al. , 2019). Despite this, mitochondrial markers have been widely used in phylogenetic studies in Neotropical deer in recent years (Gilbert et al., 2006; Duarte et al., 2008; Gutiérrez et al., 2015, 2017; Escobedo-Morales et al., 2016, 2023; Heckeberg et al., 2016, 2020; Cifuentes-Rincón et al., 2020; Peres et al., 2021a; Bernegossi et al., 2023; Sandoval et al., 2022). Besides, some studies have tested the use of nuclear markers, but they have not been informative for recovering phylogenetic relationships between lineages with recent diversification (Gilbert, Ropiquet, & Hassanin, 2006; Heckeberg, 2020; Vozdova et al. , 2021). Finally, it is worth clarifying that the PCR-RFLP protocol, which in theory would allow identifying samples of Mazama species from the Atlantic Forest (González et al. , 2009), was not able to perform species-level identification in this study because PERD samples have a mutation at the enzyme cleavage site that would discriminate M. jucunda from M. nana . The same mutation was also observed in samples previously identified as M. jucunda , for example the animal T401 from Campo Alegre, SC (Table S2), and the samples PEL34 and PNSI4 from Lauráceas State Park, PR, and Serra do Itajaí National Park, SC, respectively (Table S3). Such mutation had already been reported (Grotta-Neto, 2020) and warns us to the necessary caution when applying the PCR-RFLP method for species-specific identification of samples, considering that this approach is limited to the reference samples that it used as a comparative basis to establish the enzymatic cleavage sites. Further work should review this protocol and include a more representative group of reference samples and new enzyme sites. Conclusion remarks and Conservation implications In this work, we report a new population of the Small Red Brocket deer ( Mazama jucunda ) in the Rio Doce State Park (PERD), marking the first record for the state of Minas Gerais, located more than 700 km away from the current geographical distribution of the species (Vogliotti, Oliveira, & Duarte, 2016; Duarte et al. , 2017). However, the genetic structure observed in the analyses, along with the geographic isolation of PERD, clearly suggests that this population represents an evolutionary significant unit (ESU), and there is a possibility that it is indeed a distinct species. Despite some limitations, the present study represents an important advance in the knowledge of the geographical distribution of Neotropical forest deer species, especially regarding M. jucunda , the largest endemic animal species in the Atlantic Forest, which is one of the main biodiversity hotspot globally (Myers et al. , 2000). In the short term, the most pressing conservation implication for the species is likely to be a change in the assessment of extinction risk. Currently, M. jucunda is classified as Vulnerable by the IUCN according to criterion C2aii (Vogliotti, Oliveira, & Duarte, 2016), which means that the species has an estimated population of less than 10,000 individuals, facing declining, and with all mature individuals in a subpopulation (IUCN, 2012). Including the PERD population, the species has two subpopulations and may has an increase in population size, thus, M. jucunda would no longer be Vulnerable. Meanwhile, due to its ESU status, management actions should be customized for this population separately from the population in the south. Future research should aim to capture an individual to perform a thorough characterization analyzing morphological data, advancing molecular analyses, and, most importantly, including cytogenetic data. 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Italian Journal of Zoology 81 , 136–143. Zhang, J., Kapli, P., Pavlidis, P., & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29 , 2869–2876. Statements and Declarations Funding. This work was supported by São Paulo Research Foundation (FAPESP; n° 2021/02087-2 and 2017/07014-8) and the Coordination for the Improvement of Higher Education Personnel (CAPES; n° 88887.597519/2021-00). Competing Interests. The authors declare no competing interests. Author Contributions. Jeferson L. S. Freitas and José M. B. Duarte conceived and designed the study. Data collection was performed by Jeferson L. S. Freitas, Pedro H. F. Peres, Francisco Grotta-Neto, and Márcio L. Oliveira. Analysis was performed by Jeferson L. S. Freitas and Pedro H. F. Peres. The first draft of the manuscript was written by Jeferson L. S. Freitas, and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript. José M. B. Duarte managed and supervised the whole study. Data Availability. The data produced in this study is available in GenBank. The access codes are listed in the supplementary information Table S2 and S3. Ethics Approval. All data collection in this study was approved by the competent environmental agencies according to authorizations IEF-MG n° 015/2021 and SISBIO n° 77907. Supplementary Material File (high_resolution_images.rar) Download 78.86 MB Information & Authors Information Version history V1 Version 1 05 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Animal Genetics Keywords atlantic forest brocket deer evolutionary significant unit fecal dna Authors Affiliations Jeferson L. S. Freitas 0000-0002-7491-3553 Universidade Estadual Paulista Julio de Mesquita Filho - Campus de Jaboticabal View all articles by this author Pedro H. F. Peres Universidade Estadual Paulista Julio de Mesquita Filho - Campus de Jaboticabal View all articles by this author Francisco Grotta-Neto Universidade Estadual Paulista Julio de Mesquita Filho - Campus de Jaboticabal View all articles by this author Márcio L. Oliveira Universidade de Araraquara View all articles by this author Jose Mauricio Barbanti Duarte [email protected] Universidade Estadual Paulista Julio de Mesquita Filho - Campus de Jaboticabal View all articles by this author Metrics & Citations Metrics Article Usage 369 views 245 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jeferson L. S. Freitas, Pedro H. F. Peres, Francisco Grotta-Neto, et al. Fecal DNA reveals wider distribution and a new Evolutionary Significant Unit for an endemic deer of the Atlantic Forest (Mazama jucunda). Authorea . 05 May 2025. 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