Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Revalidation of Passalites Gloger, 1841 for the Amazon brown brocket deer P.nemorivagus (Cuvier, 1817) (Mammalia, Artiodactyla, Cervidae).

Mazamanemorivaga (Cuvier, 1817) is a gray brocket deer that inhabits the Amazon region. An assessment of previous studies revealed inconsistencies in its current taxonomic classification, suggesting the need for an update in its genus classification. A taxonomic repositioning of this species is proposed through the collection of a specimen from its type locality (French Guiana) with subsequent morphological (coloring pattern, body measurements, and craniometry), cytogenetics (G Band, C Band, conventional Giemsa, Ag-NOR staining, and BAC probe mapping), and molecular phylogenetic analysis (mitochondrial genes Cyt B of 920 bp, COI I of 658 bp, D-loop 610 bp), and comparisons with other specimens of the same taxon, as well as other Neotropical deer species. The morphological and cytogenetic differences between this and other Neotropical Cervidae confirm the taxon as a unique and valid species. The phylogenetic analysis evidenced the basal position of the M.nemorivaga specimens within the Blastocerina clade. This shows early diversification and wide divergence from the other species, suggesting that the taxon should be transferred to a different genus. A taxonomic update of the genus name is proposed through the validation of Passalites Gloger, 1841, with Passalitesnemorivagus (Cuvier, 1817) as the type species. Future research should focus on evaluating the potential existence of other species within the genus Passalites, as suggested in the literature.

Cytogenetic Mapping of Cattle BAC Probes for the Hypothetical Ancestral Karyotype of the Family Cervidae.

Cervids are characterized by their greatest karyotypic diversity among mammals. A great diversity of chromosome numbers in notably similar morphological groups leads to the existence of several complexes of cryptic species and taxonomic uncertainties. Some deer lineages, such as those of Neotropical deer, stand out for a rapid chromosomal reorganization and intraspecific chromosome polymorphisms, which have not been properly explored yet. For that reason, we contribute to the study of deer karyotype diversity and taxonomy by producing and characterizing new molecular cytogenetic markers for the gray brocket deer (Subulo gouazoubira), a deer species that retained the hypothetical ancestral karyotype of Cervidae. We used bacterial artificial chromosome (BAC) clones derived from the cattle genome (Bos taurus) as markers, which were hybridized on S. gouazoubira metaphase chromosomes. In total, we mapped 108 markers, encompassing all gray brocket deer chromosomes, except the Y chromosome. The detailed analysis of fluorescent in situ hybridization results showed 6 fissions and 1 fusion as interchromosomal rearrangements that have separated cattle and gray brocket deer karyotypes. Each group of BAC probes derived from bovine chromosome pairs 1, 2, 5, 6, 8, and 9 showed hybridization signals on 2 different chromosomes, while pairs 28 and 26 are fused in tandem in a single acrocentric chromosome in S. gouazoubira. Furthermore, the BAC markers detected the occurrence of intrachromosomal rearrangements in the S. gouazoubira chromosomes homologous to pair 1 and the X chromosome of cattle. We present a karyotypic map of the 108 new markers, which will be of great importance for future karyotypic evolution studies in cervids and, consequently, help in their conservation and taxonomy resolution.

Revalidation of Mazama rufa (Illiger 1815) (Artiodactyla: Cervidae) as a Distinct Species out of the Complex Mazama americana (Erxleben 1777).

The red brocket deer Mazama americana Erxleben, 1777 is considered a polyphyletic complex of cryptic species with wide chromosomal divergence. Evidence indicates that the observed chromosomal divergences result in reproductive isolation. The description of a neotype for M. americana allowed its genetic characterization and represented a comparative basis to resolve the taxonomic uncertainties of the group. Thus, we designated a neotype for the synonym Mazama rufa Illiger, 1815 and tested its recognition as a distinct species from the M. americana complex with the analysis of morphological, cytogenetic and molecular data. We also evaluated its distribution by sampling fecal DNA in the wild. Morphological data from craniometry and body biometry indicated an overlap of quantitative measurements between M. rufa and the entire M. americana complex. The phylogenetic hypothesis obtained through mtDNA confirmed the reciprocal monophyly relationship between M. americana and M. rufa, and both were identified as distinct molecular operational taxonomic units by the General Mixed Yule Coalescent species delimitation analysis. Finally, classic cytogenetic data and fluorescence in situ hybridization with whole chromosome painting probes showed M. rufa with a karyotype of 2n = 52, FN = 56. Comparative analysis indicate that at least fifteen rearrangements separate M. rufa and M. americana (sensu stricto) karyotypes, which confirmed their substantial chromosomal divergence. This divergence should represent an important reproductive barrier and allow its characterization as a distinct and valid species. Genetic analysis of fecal samples demonstrated a wide distribution of M. rufa in the South American continent through the Atlantic Forest, Cerrado and south region of Amazon. Thus, we conclude for the revalidation of M. rufa as a distinct species under the concept of biological isolation, with its karyotype as the main diagnostic character. The present work serves as a basis for the taxonomic review of the M. americana complex, which should be mainly based on cytogenetic characterization and directed towards a better sampling of the Amazon region, the evaluation of available names in the species synonymy and a multi-locus phylogenetic analysis.

Chromosomal Polymorphism and Speciation: The Case of the Genus Mazama (Cetartiodactyla; Cervidae).

Chromosomal polymorphism plays a major role in speciation processes in mammals with high rates of karyotypic evolution, as observed in the family Cervidae. One remarkable example is the genus Mazama that comprises wide inter- and intra-specific chromosomal variability. To evaluate the impact of chromosomal polymorphisms as reproductive barriers within the genus Mazama, inter-specific hybrids between Mazama gouazoubira and Mazama nemorivaga (MGO × MNE) and intra-specific hybrids between cytotypes of Mazama americana (MAM) differing by a tandem (TF) or centric fusion (Robertsonian translocations-RT) were evaluated. MGO × MNE hybrid fertility was evaluated by the seminal quality and testicular histology. MAM hybrids estimation of the meiotic segregation products was performed by sperm-FISH analysis. MGO × MNE hybrids analyses showed different degrees of fertility reduction, from severe subfertility to complete sterility. Regarding MAM, RT, and TF carriers showed a mean value for alternate segregation rate of 97.74%, and 67.23%, and adjacent segregation rate of 1.80%, and 29.07%, respectively. Our results suggested an efficient post-zygotic barrier represented by severe fertility reduction for MGO × MNE and MAM with heterozygous TF. Nevertheless, RT did not show a severe effect on the reproductive fitness in MAM. Our data support the validity of MGO and MNE as different species and reveals cryptic species within MAM.

Satellite DNA in Neotropical Deer Species.

The taxonomy and phylogenetics of Neotropical deer have been mostly based on morphological criteria and needs a critical revision on the basis of new molecular and cytogenetic markers. In this study, we used the variation in the sequence, copy number, and chromosome localization of satellite I-IV DNA to evaluate evolutionary relationships among eight Neotropical deer species. Using FISH with satI-IV probes derived from Mazama gouazoubira, we proved the presence of satellite DNA blocks in peri/centromeric regions of all analyzed deer. Satellite DNA was also detected in the interstitial chromosome regions of species of the genus Mazama with highly reduced chromosome numbers. In contrast to Blastocerus dichotomus, Ozotoceros bezoarticus, and Odocoileus virginianus, Mazama species showed high abundance of satIV DNA by FISH. The phylogenetic analysis of the satellite DNA showed close relationships between O. bezoarticus and B. dichotomus. Furthermore, the Neotropical and Nearctic populations of O. virginianus formed a single clade. However, the satellite DNA phylogeny did not allow resolving the relationships within the genus Mazama. The high abundance of the satellite DNA in centromeres probably contributes to the formation of chromosomal rearrangements, thus leading to a fast and ongoing speciation in this genus, which has not yet been reflected in the satellite DNA sequence diversification.

Evaluation of collared anteaters (Tamandua tetradactyla) presented in a wildlife health reference center of São Paulo state, Brazil / Avaliação de tamanduás-mirins (Tamandua tetradactyla) atendidos em um centro de referência de animais selvagens do Estado de São Paulo, Brasil

Abstract Deforestation and road construction have inflicted negative effects on wildlife populations. To contribute to the comprehension and preservation of wildlife species, this study aimed to evaluate collared anteater presented to a reference center located in São Paulo state, Brazil. Furthermore, the locations where the animals were most frequently found were determined. Data on collared anteaters (Tamandua tetradactyla) examined at the Center for Medicine and Research in Wild Animals (CEMPAS) were evaluated retrospectively for a period of 13 years. A total of 46 collared anteaters was examined, comprised of 14 males, 10 females, and 22 without identifying information. On average, adult males weigh 4.41 kg while adult females weigh 4.95 kg. The most common admission circumstance was vehicular strikes (36.96%), followed by dog bites (21.74%); 23.91% were considered clinically healthy. Animal outcomes were 36.96% (n=17) reintroduced, 34.78% (n=16) fatalities, 15.22% (n=7) transferred to other institutions, and 6.52% (n=3) escaped from the enclosure; and 6.52% (n=3) presented no medical record information. The collared anteaters were found mainly in the municipal areas of Botucatu (21.73%), São Manuel (17.39%), Lençóis Paulista (6.52%), and Bauru (4.34%), with SP-300 being the main highway associated with the occurrences. In conclusion, the main cause of lesion in the collared anteaters evaluated in this study was vehicular strike that that led to death. However, including all collared anteaters at least half of them had a positive outcome.

Resumo O desmatamento e a construção de estradas têm promovido uma série de problemas à fauna. Visando contribuir com informações que podem ser usadas na compreensão e preservação das espécies, o trabalho teve por objetivo avaliar tamanduás-mirins atendidos em um centro de referência localizado no Estado de São Paulo, Brazil. Foram avaliados retrospectivamente dados referentes a tamanduás-mirins (Tamandua tetradactyla), que foram trazidos ao Centro de Medicina e Pesquisa em Animais Selvagens (CEMPAS), durante um período de 13 anos. De um total de 46 tamanduás-mirins atendidos, 14 foram machos e 10 foram fêmeas, e 22 não possuíam identificação quanto ao sexo. Em média, os machos adultos pesaram 4,41 kg, enquanto as fêmeas adultas pesaram 4,95 kg. Com relação à circunstância que levou ao atendimento, as mais importantes foram o atropelamento (36,96%) e a mordedura de cães (21,74%), sendo que 23,91% foram considerados clinicamente saudáveis. No tocante à evolução dos casos, 36,96% (n=17) foram reintroduzidos, 34.78% (n=16) foram a óbito, 15,22% (n=7) foram transferidos para outras instituições, 6,52% (n=3) evadiram do recinto e 6,52% (n=3) não há informação. Os animais foram provenientes principalmente dos municípios de Botucatu (21,73%), São Manuel (17,39%), Lençóis Paulista (6,52%) e Bauru (4,34%), sendo a SP-300 a principal rodovia relacionada as ocorrências. Foi possível concluir que dos tamanduás-mirins avaliados a principal causa de lesão foi o atropelamento, que teve por evolução o óbito; porém ao se incluir os tamanduás-mirins saudáveis e demais, ao menos metade evoluíram positivamente.