Ex Situ Approaches for the Conservation of Genetic Resources in the Superorder Xenarthra.

Xenarthra-a superorder of placental mammals endemic to the Neotropics-is represented by armadillos, anteaters, and sloths. Considering their long history in the Americas, extant xenarthrans represent an important group for understanding the impact of past environmental changes on species diversification and serve key ecological functions as ecosystem engineers. Unfortunately, most wild xenarthran populations are at risk, due primarily to anthropogenic activities, necessitating urgent conservation efforts. Moreover, the paucity of information on some species has rendered population estimation and, consequently, conservation management challenging. In addition, relatively few groups are researching this superorder, perhaps because fieldwork with armadillos, anteaters, or sloths and their captive care are challenging tasks. Nevertheless, dedicated research and efforts to ensure the long-term conservation of these animals are deemed essential. In this context, cryobanks are a practical approach for breeding and maintaining genetic diversity in wildlife, and they are important tools for assisting and improving both ex situ and in situ conservation strategies. Therefore, cryopreservation of biological resources may be a promising strategy for conserving xenarthrans. Specifically, semen cryopreservation, which has already been applied in some species, may be the most effective strategy for this group. The present article provides an overview of ex situ conservation of xenarthrans, which will contribute to the development and implementation of additional strategies for protecting these unique mammals.

Resolving the phylogenetic position of Darwin's extinct ground sloth (Mylodon darwinii) using mitogenomic and nuclear exon data.

Mylodon darwinii is the extinct giant ground sloth named after Charles Darwin, who first collected its remains in South America. We have successfully obtained a high-quality mitochondrial genome at 99-fold coverage using an Illumina shotgun sequencing of a 12 880-year-old bone fragment from Mylodon Cave in Chile. Low level of DNA damage showed that this sample was exceptionally well preserved for an ancient subfossil, probably the result of the dry and cold conditions prevailing within the cave. Accordingly, taxonomic assessment of our shotgun metagenomic data showed a very high percentage of endogenous DNA with 22% of the assembled metagenomic contigs assigned to Xenarthra. Additionally, we enriched over 15 kb of sequence data from seven nuclear exons, using target sequence capture designed against a wide xenarthran dataset. Phylogenetic and dating analyses of the mitogenomic dataset including all extant species of xenarthrans and the assembled nuclear supermatrix unambiguously place Mylodon darwinii as the sister-group of modern two-fingered sloths, from which it diverged around 22 million years ago. These congruent results from both the mitochondrial and nuclear data support the diphyly of the two modern sloth lineages, implying the convergent evolution of their unique suspensory behaviour as an adaption to arboreality. Our results offer promising perspectives for whole-genome sequencing of this emblematic extinct taxon.

Genotoxic effects of Roundup Full II® on lymphocytes of Chaetophractus villosus (Xenarthra, Mammalia): In vitro studies.

In Argentina, Chaetophractus villosus has a wide distribution that overlaps with agricultural areas where soybean is the predominant crop. In such areas the pesticide Roundup Full II® (RU) is widely applied. The genotoxic effect of its active ingredient glyphosate (RU is 66.2% glyphosate) on the peripheral blood lymphocytes of C. villosus was tested over a range of concentrations (280, 420, 560, 1120 µmol/L). Culture medium without glyphosate served as negative control, while medium containing mitomycin C served as positive control. Genetic damage was characterized in terms of the percentage of cells with chromosome aberrations (CA), the mean number of sister chromatid exchanges (SCE) per cell, and the modification of cell proliferation kinetics via the calculation of the replication index. Significant increases (p < 0.0001) were seen in the CA frequency and the mean number of SCEs per cell compared to negative controls at all the RU concentrations tested. Chromatid breaks, the only form of CA observed, under the 560 µmol/L RU conditions and in presence of mitomycin C were four to five times more common than at lower concentrations, while no viable cells were seen in the 1120 µmol/L treatment. The mean number of SCEs per cell was significantly higher under the 280 µmol/L RU conditions than the 420 or 560 µmol/L RU conditions; cells cultivated in the presence of MMC also showed significantly more SCEs. All the RU concentrations tested (except in the 1120 µmol/L RU treatment [no viable cells]) induced a significant reduction in the replication index (p < 0.0001). The present results confirm the genotoxic effects of RU on C. villosus lymphocytes in vitro, strongly suggesting that exposure to RU could induce DNA damage in C. villosus wildlife.

Ancient DNA from the extinct South American giant glyptodont Doedicurus sp. (Xenarthra: Glyptodontidae) reveals that glyptodonts evolved from Eocene armadillos.

Glyptodonts were giant (some of them up to ~2400 kg), heavily armoured relatives of living armadillos, which became extinct during the Late Pleistocene/early Holocene alongside much of the South American megafauna. Although glyptodonts were an important component of Cenozoic South American faunas, their early evolution and phylogenetic affinities within the order Cingulata (armoured New World placental mammals) remain controversial. In this study, we used hybridization enrichment and high-throughput sequencing to obtain a partial mitochondrial genome from Doedicurus sp., the largest (1.5 m tall, and 4 m long) and one of the last surviving glyptodonts. Our molecular phylogenetic analyses revealed that glyptodonts fall within the diversity of living armadillos. Reanalysis of morphological data using a molecular 'backbone constraint' revealed several morphological characters that supported a close relationship between glyptodonts and the tiny extant fairy armadillos (Chlamyphorinae). This is surprising as these taxa are among the most derived cingulates: glyptodonts were generally large-bodied and heavily armoured, while the fairy armadillos are tiny (~9-17 cm) and adapted for burrowing. Calibration of our phylogeny with the first appearance of glyptodonts in the Eocene resulted in a more precise timeline for xenarthran evolution. The osteological novelties of glyptodonts and their specialization for grazing appear to have evolved rapidly during the Late Eocene to Early Miocene, coincident with global temperature decreases and a shift from wet closed forest towards drier open woodland and grassland across much of South America. This environmental change may have driven the evolution of glyptodonts, culminating in the bizarre giant forms of the Pleistocene.

Esforços para conservação da espécie Myrmecophaga tridactyla Linnaeus, 1758: tecnologias aplicadas à reprodução / Efforts for the conservation of the Myrmecophaga tridactyla species, Linnaeus, 1758: technologies applied to reproduction

Esforços para conservação do Myrmecophaga tridactyla têm sido realizados. Esta espécie sofre com adestruição de seu habitat natural, incêndios, atropelamentos nas rodovias, entre outros. A presente revisãoapresenta os esforços envidados na conservação do tamanduá-bandeira, com ênfase nas tecnologias aplicadas àreprodução.(AU)

Efforts for the conservation of the Myrmecophaga tridactyla have been done. This species suffers withthe destruction of their natural habitat, fire, road kill on highways, among others. The present review shows theefforts of conservation of the giant anteater, with emphasis on technologies applied to reproduction.(AU)

Esforços para conservação da espécie Myrmecophaga tridactyla Linnaeus, 1758: tecnologias aplicadas à reprodução / Efforts for the conservation of the Myrmecophaga tridactyla species, Linnaeus, 1758: technologies applied to reproduction

Esforços para conservação do Myrmecophaga tridactyla têm sido realizados. Esta espécie sofre com adestruição de seu habitat natural, incêndios, atropelamentos nas rodovias, entre outros. A presente revisãoapresenta os esforços envidados na conservação do tamanduá-bandeira, com ênfase nas tecnologias aplicadas àreprodução.

Efforts for the conservation of the Myrmecophaga tridactyla have been done. This species suffers withthe destruction of their natural habitat, fire, road kill on highways, among others. The present review shows theefforts of conservation of the giant anteater, with emphasis on technologies applied to reproduction.

Historical and non-invasive samples: a study case of genotyping errors in newly isolated microsatellites for the lesser anteater (Tamandua tetradactyla L., Pilosa).

Tamandua tetradactyla (Pilosa), the lesser anteater, is a medium-size mammal from South America. Its wide distribution through different landscapes, solitary and nocturnal habits, and the difficulty to capture and contain specimens limit the amount of individuals and populations sampled during fieldworks. These features along with the lack of specific molecular markers for the lesser anteater might be the causes for paucity in population genetic studies for the species. Historical samples from museum specimens, such as skins, and non-invasive samples, such as plucked hair, can be supplementary sources of DNA samples. However, the DNA quantity and quality of these samples may be limiting factors in molecular studies. In this study, we describe nine microsatellite loci for T. tetradactyla and test the amplification success, data reliability and estimate errors on both historical and non-invasive sample sets. We tested nine polymorphic microsatellites and applied the quality index approach to evaluate the relative performance in genotype analysis of 138 historical samples (study skin) and 19 non-invasive samples (plucked hair). The observed results show a much superior DNA quality of non-invasive over historical samples and support the quality index analysis as a practical tool to exclude samples with doubtful performance in genetic studies. We also found a relationship between the age of non-invasive samples and DNA quality, but lack of evidence of this pattern for historical samples.

Making the impossible possible: rooting the tree of placental mammals.

Untangling the root of the evolutionary tree of placental mammals has been nearly an impossible task. The good news is that only three possibilities are seriously considered. The bad news is that all three possibilities are seriously considered. Paleontologists favor a root anchored by Xenarthra (e.g., sloths and anteater), whereas molecular evolutionists have favored the two other possible roots: Afrotheria (e.g., elephants, hyraxes, and tenrecs) and Atlantogenata (Afrotheria + Xenarthra). Now, two groups of researchers have scrutinized the largest available genomic data sets bearing on the question and have come to opposite conclusions, as reported in this issue of Molecular Biology and Evolution. Needless to say, more research is needed.

A new species of Neosclerocalyptus Paula Couto (Mammalia: Xenarthra: Cingulata): the oldest record of the genus and morphological and phylogenetic aspects.

Among South American Quaternary Glyptodontidae (Mammalia, Cingulata), Neosclerocalyptus Paula Couto represents one of the best known genera. Prior to this contribution, four species were recognized. N. pseudornatus (Ameghino) and N. ornatus (Owen) (Ensenadan Age/Stage, early-middle Pleistocene); N. gouldi Zurita (Bonaerian Age/Stage, middle Pleistocene-late Pleistocene), and N. paskoensis (Zurita) (Lujanian Age/Stage, late Pleistocene-early Holocene). One of the most notable characters of the species of the genus is a modified area located in the distal part of the nasals, recently interpreted as a neomorphic structure derived from the ossification of the nasal cartilages. In this contribution, a new species of Neosclerocalyptus (N. castellanosi sp. nov.), which in turn represents the oldest record of the genus, is presented and described. In addition, a cladistic analysis is carried out to test the monophyly of Neosclerocalyptus and the phylogenetic position of this new species. The material comes from Vorohuean (late Pliocene) levels in the surroundings of Mar del Plata, Buenos Aires Province, Argentina. Among other morphological characters, this new species has ossified nasal cartilages restricted to the latero-dorsal area of the nasals, whereas in the remaining species these structures are more expanded and both sides contact in the midline of the skull. In turn, the phylogenetic analysis confirmed the monophyly of Neosclerocalyptus, whereas N. castellanosi sp. nov. appears closely related to N. pseudornatus, being N. ornatus the sister taxa of this clade. On the other hand, N. gouldi + N. paskoensis constitute the other clade. The clade constituted by Hoplophorus euphractus Lund + Panochthus intermedius Lydekker constitutes the sister taxa of Neosclerocalyptus.

The chromosomes of Afrotheria and their bearing on mammalian genome evolution.

Afrotheria is the clade of placental mammals that, together with Xenarthra, Euarchontoglires and Laurasiatheria, represents 1 of the 4 main recognized supraordinal eutherian clades. It reunites 6 orders of African origin: Proboscidea, Sirenia, Hyracoidea, Macroscelidea, Afrosoricida and Tubulidentata. The apparently unlikely relationship among such disparate morphological taxa and their possible basal position at the base of the eutherian phylogenetic tree led to a great deal of attention and research on the group. The use of biomolecular data was pivotal in Afrotheria studies, as they were the basis for the recognition of this clade. Although morphological evidence is still scarce, a plethora of molecular data firmly attests to the phylogenetic relationship among these mammals of African origin. Modern cytogenetic techniques also gave a significant contribution to the study of Afrotheria, revealing chromosome signatures for the group as a whole, as well as for some of its internal relationships. The associations of human chromosomes HSA1/19 and 5/21 were found to be chromosome signatures for the group and provided further support for Afrotheria. Additional chromosome synapomorphies were also identified linking elephants and manatees in Tethytheria (the associations HSA2/3, 3/13, 8/22, 18/19 and the lack of HSA4/8) and elephant shrews with the aardvark (HSA2/8, 3/20 and 10/17). Herein, we review the current knowledge on Afrotheria chromosomes and genome evolution. The already available data on the group suggests that further work on this apparently bizarre assemblage of mammals will provide important data to a better understanding on mammalian genome evolution.

Chromosome evolution in Xenarthra: new insights from an ancient group.

The Magnaorder Xenarthra is one of the four main supraordinal eutherian clades, together with Afrotheria, Euarchontoglires and Laurasiatheria. Xenarthra is an eminently Central and South American group of special interest in phylogenetic studies due to its possible position at the base of the eutherian tree. The use of modern cytogenetic techniques in some species of Xenarthra has provided important insights into the karyotypic evolution of mammals. Nevertheless, chromosome analyses in the group are still restricted, with only a few individuals of each species studied and karyotype descriptions mostly without banding patterns. In addition, it is likely that still unknown species exist and that the chromosome variability in the group is underestimated. We present a review of the currently available data on Xenarthra chromosomes and genomes and on the impact that their study has had in the understanding of mammalian genome evolution. It is clear that further cytogenetic analyses in Xenarthra, including banding patterns and molecular approaches, are likely to help in the identification of new species, reveal still undetected chromosome variations, provide information to support conservation strategies planning, and greatly contribute to a better understanding of mammalian genome evolution.

Oldest cingulate skulls provide congruence between morphological and molecular scenarios of armadillo evolution.

The cingulates of the mammalian order Xenarthra present a typical case of disagreement between molecular and morphological phylogenetic studies. We report here the discovery of two new skulls from the Late Oligocene Salla Beds of Bolivia (approx. 26 Ma), which are the oldest known well-preserved cranial remains of the group. A new taxon is described: Kuntinaru boliviensis gen. et sp. nov. A phylogenetic analysis clusters K. boliviensis together with the armadillo subfamily Tolypeutinae. These skulls document an early spotty occurrence for the Tolypeutinae at 26 Ma, in agreement with the temporal predictions of previous molecular studies. The fossil record of tolypeutines is now characterized by a unique occurrence in the Late Oligocene, and a subsequent 12 Myr lack in the fossil record. It is noteworthy that the tolypeutines remain decidedly marginal in the Late Palaeogene and Early Neogene deposits, whereas other cingulate groups diversify. Also, the anatomical phylogenetic analysis herein, which includes K. boliviensis, is congruent with recent molecular phylogenetic analyses. Kuntinaru boliviensis is the oldest confident calibration point available for the whole Cingulata.

The karyotype of Cabassous unicinctus (Dasypodidae, Xenarthra)

Abstract ­ Armadillos was belonged to the Xenarthra Order, Dasypodidae family. This family has been comprising the largest number of genera and species among the Xenarthrans; eight (8) and 21, respectively. Two adult males of the species Cabassous unicinctus were analyzed in this study. Lymphocyte cultures from whole blood were used and the cells were then submitted to conventional staining by C- and Ag-NOR banding. Data regarding the number of chromosomes showed discrepancies among the species described in the literature. The 46 chromosomes observed were distributed in six large metacentric pairs, five medium submetacentric pairs, five medium and small metacentric pairs and six acrocentric pairs. The Y chromosome was classified such as the smallest acrocentric of the group. The X chromosome was classified as medium submetacentric, it considered atypical for mammals, because X was generally large size metacentric when it compared to the remaining karyotype. The diploid number reduction from 62 to 46 chromosomes may be explained by Robertsonian fusion and the inversion of acrocentric, or even the fission of centromeric regions of metacentric, given that the species Cabassous centralis had 23 acrocentric pairs and that this number was reduced to 14 pairs in the Cabassous tautouay and six pairs in the species described in the present work.

Synaptonemal complexes and XY behavior in two species of argentinian armadillos: Chaetophractus villosus and Dasypus hybridus (Xenarthra, Dasypodidae)

Spermatocytes from the two armadillo species, C. villosus and D. hybridus were studied in microspreads for synaptonemal complexes (SCs) and in thin sections for electron microscopy (EM). The complete se karyotype generally agrees with previous reports on mitotic chromosomes, except for the sex chromosomes. The X chromosome is submetacentric in both species and the Y is the shortest one in C. villosus and the second shortest in D. hybridus, and an extremely acrocentric one. A SC is formed along the total length of the Y chromosome, and this SC persists along all the pachytene substages. A single recombi-nation nodule (RN) is located in the region of the se nearest to the attachment to the nuclear envelope. The lateral element (LE) of the X axis in the SC shows a wavy aspect in most of the se length distant from the nuclear envelope. Nucleoli are attached to acrocentric or submetacentric bivalents, are visibly double in some cells , and in thin sections show an elaborate nucleolonema. Some differences in the XY are species-specific, as the higher degree of tangling and stronger heteropycnosis in D. hybridus. The effective, single crossover of the XY pair is highly localized, despite the permanence of a long tract of SC

Synaptonemal complexes and XY behavior in two species of argentinian armadillos: Chaetophractus villosus and Dasypus hybridus (Xenarthra, Dasypodidae)

Spermatocytes from the two armadillo species, C. villosus and D. hybridus were studied in microspreads for synaptonemal complexes (SCs) and in thin sections for electron microscopy (EM). The complete se karyotype generally agrees with previous reports on mitotic chromosomes, except for the sex chromosomes. The X chromosome is submetacentric in both species and the Y is the shortest one in C. villosus and the second shortest in D. hybridus, and an extremely acrocentric one. A SC is formed along the total length of the Y chromosome, and this SC persists along all the pachytene substages. A single recombi-nation nodule (RN) is located in the region of the se nearest to the attachment to the nuclear envelope. The lateral element (LE) of the X axis in the SC shows a wavy aspect in most of the se length distant from the nuclear envelope. Nucleoli are attached to acrocentric or submetacentric bivalents, are visibly double in some cells , and in thin sections show an elaborate nucleolonema. Some differences in the XY are species-specific, as the higher degree of tangling and stronger heteropycnosis in D. hybridus. The effective, single crossover of the XY pair is highly localized, despite the permanence of a long tract of SC(AU)

Synaptonemal complexes and XY behavior in two species of argentinian armadillos: Chaetophractus villosus and Dasypus hybridus (Xenarthra, Dasypodidae)

Spermatocytes from the two armadillo species, C. villosus and D. hybridus were studied in microspreads for synaptonemal complexes (SCs) and in thin sections for electron microscopy (EM). The complete se karyotype generally agrees with previous reports on mitotic chromosomes, except for the sex chromosomes. The X chromosome is submetacentric in both species and the Y is the shortest one in C. villosus and the second shortest in D. hybridus, and an extremely acrocentric one. A SC is formed along the total length of the Y chromosome, and this SC persists along all the pachytene substages. A single recombi-nation nodule (RN) is located in the region of the se nearest to the attachment to the nuclear envelope. The lateral element (LE) of the X axis in the SC shows a wavy aspect in most of the se length distant from the nuclear envelope. Nucleoli are attached to acrocentric or submetacentric bivalents, are visibly double in some cells , and in thin sections show an elaborate nucleolonema. Some differences in the XY are species-specific, as the higher degree of tangling and stronger heteropycnosis in D. hybridus. The effective, single crossover of the XY pair is highly localized, despite the permanence of a long tract of SC(AU)

Chromosomal localization of the telomeric (TTAGGG)n sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra.

The distribution of the vertebrate telomeric sequence (TTAGGG)(n) in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.

Identification of microsatellite DNA markers for the giant anteater Myrmecophaga tridactyla.

The giant anteater (Myrmecophaga tridactyla) is found from Belize and Guatemala to Paraguay and Argentina. Its conservation status is considered vulnerable by IUCN. Here we report the isolation and characterization of six microsatellite loci. Positive loci for (GT)(n) were isolated using a magnetic bead hybridization selection protocol. The number of alleles per locus as well as the heterozygosity and PCR conditions are described. These loci will be useful for studying population structure, genetic diversity, and paternity in M. tridactyla wild populations.

Influence of Tertiary paleoenvironmental changes on the diversification of South American mammals: a relaxed molecular clock study within xenarthrans.

BACKGROUND: Comparative genomic data among organisms allow the reconstruction of their phylogenies and evolutionary time scales. Molecular timings have been recently used to suggest that environmental global change have shaped the evolutionary history of diverse terrestrial organisms. Living xenarthrans (armadillos, anteaters and sloths) constitute an ideal model for studying the influence of past environmental changes on species diversification. Indeed, extant xenarthran species are relicts from an evolutionary radiation enhanced by their isolation in South America during the Tertiary era, a period for which major climate variations and tectonic events are relatively well documented. RESULTS: We applied a Bayesian approach to three nuclear genes in order to relax the molecular clock assumption while accounting for differences in evolutionary dynamics among genes and incorporating paleontological uncertainties. We obtained a molecular time scale for the evolution of extant xenarthrans and other placental mammals. Divergence time estimates provide substantial evidence for contemporaneous diversification events among independent xenarthran lineages. This correlated pattern of diversification might possibly relate to major environmental changes that occurred in South America during the Cenozoic. CONCLUSIONS: The observed synchronicity between planetary and biological events suggests that global change played a crucial role in shaping the evolutionary history of extant xenarthrans. Our findings open ways to test this hypothesis further in other South American mammalian endemics like hystricognath rodents, platyrrhine primates, and didelphid marsupials.

Mitotic and meiotic chromosome studies in silky anteater Cyclopes didactylus (Myrmecophagidae: Xenarthra).

The karyotype of a male pigmy anteater, Cyclopes didactylus, an endangered species from the Amazon region, is described. The size and morphology of the X and Y chromosomes in mitotic and meiotic analyses is recorded and discussed.