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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.

The risk of developing cancer is correlated with body size and lifespan within species, but there is no correlation between cancer and either body size or lifespan between species indicating that large, long-lived species have evolved enhanced cancer protection mechanisms. Previously we showed that several large bodied Afrotherian lineages evolved reduced intrinsic cancer risk, particularly elephants and their extinct relatives ( Proboscideans ), coincident with pervasive duplication of tumor suppressor genes (Vazquez and Lynch, 2021). Unexpectedly, we also found that Xenarthrans (sloths, armadillos, and anteaters) evolved very low intrinsic cancer risk. Here, we show that: (1) several Xenarthran lineages independently evolved large bodies, long lifespans, and reduced intrinsic cancer risk; (2) the reduced cancer risk in the stem lineages of Xenarthra and Pilosa coincided with bursts of tumor suppressor gene duplications; (3) cells from sloths proliferate extremely slowly while Xenarthran cells induce apoptosis at very low doses of DNA damaging agents; and (4) the prevalence of cancer is extremely low Xenarthrans , and cancer is nearly absent from armadillos. These data implicate the duplication of tumor suppressor genes in the evolution of remarkably large body sizes and decreased cancer risk in Xenarthrans and suggest they are a remarkably cancer-resistant group of mammals., Competing Interests: JV, MP, BM, MK, LA, VL No competing interests declared

Fossil sloths are regarded as obligate herbivores for reasons including peculiarities of their craniodental morphology and that all living sloths feed exclusively on plants. We challenge this view based on isotopic analyses of nitrogen of specific amino acids, which show that Darwin's ground sloth Mylodon darwinii was an opportunistic omnivore. This direct evidence of omnivory in an ancient sloth requires reevaluation of the ecological structure of South American Cenozoic mammalian communities, as sloths represented a major component of these ecosystems across the past 34 Myr. Furthermore, by analyzing modern mammals with known diets, we provide a basis for reliable interpretation of nitrogen isotopes of amino acids of fossils. We argue that a widely used equation to determine trophic position is unnecessary, and that the relative isotopic values of the amino acids glutamate and phenylalanine alone permit reliable reconstructions of trophic positions of extant and extinct mammals., (© 2021. The Author(s).)

Pangolins are a group of eight endangered mammalian species included in the family Manidae (Order Pholidota) and divided into four each African (Arboreal species; Phataginus tetradactyla, Phataginus tricuspis, Ground dwelling species; Smutsia gigantea and Smutsia temminckii) and Asian (Ground dwelling; Manis crassicaudata, Manis pentadactyla, Manis javanica and Manis culionensis) species. The taxonomy of all the eight extant pangolin species based on molecular genetics studies, remains unresolved and poorly examined. The present study is to address this lacuna by studying the phylogenetic, taxonomic status and molecular dating of Indian pangolin with other six out of eight extant pangolins (Sunda pangolin possibly extinct) based on complete coding region of mitochondrial cytochrome b gene. Overall sequences divergence among pangolins ranged between 0.01 ± 0.01 and 0.26 ± 0.03, where within 'Manis' it ranged between 0.01 ± 0.01 and 0.14 ± 0.03. Cytochrome b sequences based phylogenetic tree revealed, the division of seven pangolin species into two paraphyletic clades of African and Asian species, further these two paraphyletic clades were divided into three well-supported monophyletic clades, first for the genus 'Smutsia' with two African ground pangolins, second for the genus 'Phataginus' with two African arboreal pangolins and third for 'Manis' with three Asian species. Within clade of 'Manis', Chinese pangolin and Malayan pangolin are basal where Indian pangolin is present as a sister clade and furthermore, molecular dating analysis suggested that pangolins diverged from Carnivora at ∼87.2 MYA, followed by the split of Asian pangolins and African pangolins at ∼36.1 MYA and Indian pangolin split from Chinese pangolin and Malayan pangolin at ∼16.7 MYA.

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., (© 2018 The Authors.)

Pangolins, considered the most-trafficked mammals on Earth, are rapidly heading to extinction. Eight extant species of these African and Asian scale-bodied anteaters are commonly recognized, but their evolutionary relationships remain largely unexplored. Here, we present the most comprehensive phylogenetic assessment of pangolins, based on genetic variation of complete mitogenomes and 9 nuclear genes. We confirm deep divergence among Asian and African pangolins occurring not later than the Oligocene-Miocene boundary ca. 23 million years ago (Ma) (95% HPD = 18.7-27.2), limited fossil evidence suggesting dispersals from Europe. We recognize 3 genera including Manis (Asian pangolins), Smutsia (large African pangolins), and Phataginus (small African pangolins), which first diversified in the Middle-Upper Miocene (9.8-13.3 Ma) through a period of gradual cooling coinciding with a worldwide taxonomic diversification among mammals. Based on large mitogenomic distances among the 3 genera (18.3-22.8%) and numerous (18) morphological traits unique to Phataginus, we propose the subfamily Phatagininae subfam. nov. to designate small African pangolins. In contrast with the morphological-based literature, our results establish that the thick-tailed pangolin (Manis crassicaudata) is sister-species of the Sunda (Manis javanica) and Palawan (Manis culionensis) pangolins. Mitogenomic phylogenetic delineations supported additional pangolin species subdivisions (n = 13), including 6 African common pangolin (Phataginus tricuspis) lineages, but these patterns were not fully supported by our multi-locus approach. Finally, we identified more than 5000 informative mitogenomic sites and diagnostic variation from 5 nuclear genes among all species and lineages of pangolins, providing an important resource for further research and for effectively tracing the worldwide pangolin trade.

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.

Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother-offspring dyads of Hoffman's two-toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference-aligned and de novo-assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo-assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference-aligned and de novo-assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference-aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo-assembled data sets. We observed approximately 10- and 13-fold declines in the number of loci sampled in the reference-aligned and de novo-assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade-off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction-enzyme-based SNP studies., (© 2016 John Wiley & Sons Ltd.)

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., (© 2016 John Wiley & Sons Ltd.)

Xenarthra (armadillos, sloths, and anteaters) constitutes one of the four major clades of placental mammals. Despite their phylogenetic distinctiveness in mammals, a reference phylogeny is still lacking for the 31 described species. Here we used Illumina shotgun sequencing to assemble 33 new complete mitochondrial genomes, establishing Xenarthra as the first major placental clade to be fully sequenced at the species level for mitogenomes. The resulting data set allowed the reconstruction of a robust phylogenetic framework and timescale that are consistent with previous studies conducted at the genus level using nuclear genes. Incorporating the full species diversity of extant xenarthrans points to a number of inconsistencies in xenarthran systematics and species definition. We propose to split armadillos into two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 Ma. Species delimitation within long-nosed armadillos (genus Dasypus) appeared more complex than anticipated, with the discovery of a divergent lineage in French Guiana. Diversification analyses showed Xenarthra to be an ancient clade with a constant diversification rate through time with a species turnover driven by high but constant extinction. We also detected a significant negative correlation between speciation rate and past temperature fluctuations with an increase in speciation rate corresponding to the general cooling observed during the last 15 My. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guiana Shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Aardvark (Orycteropus afer) is a very unique, but relatively widespread African mammal. Although some morphological variation has been observed between forest and savannah populations and among different African regions, they are all considered as a single species. However, no modern taxonomic revision is available. All captive aardvarks in Europe are believed to stem from wild born animals from Namibia, but recently several new wild-caught aardvarks from Tanzania have been integrated into the captive population. This raises the question, whether these specimens should be interbred with the existing captive population or whether there is a risk of outbreeding depression. We studied the genetic structure of the captive populations by sequencing two mitochondrial genes (cytochrome b and 16S rRNA) to assess the degree of genetic differentiation between the two source regions. Our data suggest that the aardvarks kept in European zoos belong to the same phylogenetic (mitochondrial) lineage as the differentiation in the two studied mitochondrial markers was extremely low. A more comprehensive analysis of a larger sample with well documented origin (covering the complete geographic range) and with more sensitive genetic markers is needed to infer any final conclusions concerning the aardvark's taxonomy and identification of suitable aardvark management units., (© 2014 Wiley Periodicals, Inc.)

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., (© 2013 John Wiley & Sons Ltd.)

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.

Bone microstructure reflects physiological characteristics and has been shown to contain phylogenetic and ecological signals. Although mammalian long bone histology is receiving increasing attention, systematic examination of the main clades has not yet been performed. Here we describe the long bone microstructure of Xenarthra based on thin sections representing twenty-two species. Additionally, patterns in bone compactness of humeri and femora are investigated. The primary bone tissue of xenarthran long bones is composed of a mixture of woven, parallel-fibered and lamellar bone. The vascular canals have a longitudinal, reticular or radial orientation and are mostly arranged in an irregular manner. Concentric rows of vascular canals and laminar organization of the tissue are only found in anteater bones. The long bones of adult specimens are marked by dense Haversian bone, a feature that has been noted for most groups of mammals. In the long bones of armadillos, secondary osteons have an oblique orientation within the three-dimensional bone tissue, thus resulting in their irregular shape when the bones are sectioned transversely. Secondary remodeling is generally more extensive in large taxa than in small taxa, and this could be caused by increased loading. Lines of arrested growth are assumed to be present in all specimens, but they are restricted to the outermost layer in bones of armadillos and are often masked by secondary remodeling in large taxa. Parameters of bone compactness show a pattern in the femur that separates Cingulata and Pilosa (Folivora and Vermilingua), with cingulates having a lower compactness than pilosans. In addition, cingulates show an allometric relationship between humeral and femoral bone compactness.

To discover interordinal relationships of living and fossil placental mammals and the time of origin of placentals relative to the Cretaceous-Paleogene (K-Pg) boundary, we scored 4541 phenomic characters de novo for 86 fossil and living species. Combining these data with molecular sequences, we obtained a phylogenetic tree that, when calibrated with fossils, shows that crown clade Placentalia and placental orders originated after the K-Pg boundary. Many nodes discovered using molecular data are upheld, but phenomic signals overturn molecular signals to show Sundatheria (Dermoptera + Scandentia) as the sister taxon of Primates, a close link between Proboscidea (elephants) and Sirenia (sea cows), and the monophyly of echolocating Chiroptera (bats). Our tree suggests that Placentalia first split into Xenarthra and Epitheria; extinct New World species are the oldest members of Afrotheria.

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 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., (Copyright © 2012 S. Karger AG, Basel.)

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., (Copyright © 2012 S. Karger AG, Basel.)

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., (This journal is © 2011 The Royal Society)

Previous analyses of how mammals vary in their ossification sequences have focused on monotremes, marsupials, and boreoeutherian placentals. Here, we focus on the sequence of cranial and postcranial ossification events during growth in the xenarthran skull and skeleton, including armadillos, anteaters, and sloths. We use two different methods to quantify sequence heterochrony: sequence analysis of variance (ANOVA) and event-paring/Parsimov. Our results indicate that Parsimov is conservative and does not detect clear heterochronic shifts between xenarthran and boreoeutherian placentals. Sequence-ANOVA performs better, but both methods exhibit sensitivity to the artifactual accumulation of ties. By controlling for ties and taking into account results that the methods have in common, our analysis suggests that xenarthrans significantly differ from other placentals by a late ossification of the sternum and an early ossification of the phalanges and pubis. We interpret these differences as showing that heterochrony plays a role in the skeletal development of xenarthrans, a change from previous studies that have emphasized the developmental homogeneity of the skeleton across placental mammals., (© 2011 Wiley Periodicals, Inc.)

One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The "presence" of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its "absence" in more distant species. To resolve the placental origin controversy we extracted approximately 2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a "soft" polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy.

Interpreting the evolution of neuronal types in the cerebral cortex of mammals requires information from a diversity of species. However, there is currently a paucity of data from the Xenarthra and Afrotheria, two major phylogenetic groups that diverged close to the base of the eutherian mammal adaptive radiation. In this study, we used immunohistochemistry to examine the distribution and morphology of neocortical neurons stained for nonphosphorylated neurofilament protein, calbindin, calretinin, parvalbumin, and neuropeptide Y in three xenarthran species-the giant anteater (Myrmecophaga tridactyla), the lesser anteater (Tamandua tetradactyla), and the two-toed sloth (Choloepus didactylus)-and two afrotherian species-the rock hyrax (Procavia capensis) and the black and rufous giant elephant shrew (Rhynchocyon petersi). We also studied the distribution and morphology of astrocytes using glial fibrillary acidic protein as a marker. In all of these species, nonphosphorylated neurofilament protein-immunoreactive neurons predominated in layer V. These neurons exhibited diverse morphologies with regional variation. Specifically, high proportions of atypical neurofilament-enriched neuron classes were observed, including extraverted neurons, inverted pyramidal neurons, fusiform neurons, and other multipolar types. In addition, many projection neurons in layers II-III were found to contain calbindin. Among interneurons, parvalbumin- and calbindin-expressing cells were generally denser compared to calretinin-immunoreactive cells. We traced the evolution of certain cortical architectural traits using phylogenetic analysis. Based on our reconstruction of character evolution, we found that the living xenarthrans and afrotherians show many similarities to the stem eutherian mammal, whereas other eutherian lineages display a greater number of derived traits.

Placental (eutherian) mammals are currently classified into four superordinal clades (Afrotheria, Xenarthra, Laurasiatheria and Supraprimates) of which one, the Afrotheria (a unique lineage of African origin), is generally considered to be basal. Therefore, Afrotheria provide a pivotal evolutionary link for studying fundamental differences between the sex chromosomes of human/mouse (both representatives of Supraprimates and the index species for studies of sex chromosomes) and those of the distantly related marsupials. In this study, we use female fibroblasts to investigate classical features of X chromosome inactivation including replication timing of the X chromosomes and Barr body formation. We also examine LINE-1 accumulation on the X chromosomes of representative afrotherians and look for evidence of a pseudoautosomal region (PAR). Our results demonstrate that asynchronous replication of the X chromosomes is common to Afrotheria, as with other mammals, and Barr body formation is observed across all Placentalia, suggesting that mechanisms controlling this evolved before their radiation. Finally, we provide evidence of a PAR (which marsupials lack) and demonstrate that LINE1 is accumulated on the afrotherian and xenarthran X, although this is probably not due to transposition events in a common ancestor, but rather ongoing selection to retain recently inserted LINE1 on the X.

Armadillos, anteaters, and sloths (Order Xenarthra) comprise 1 of the 4 major clades of placental mammals. Isolated in South America from the other continental landmasses, xenarthrans diverged over a period of about 65 Myr, leaving more than 200 extinct genera and only 31 living species. The presence of both ancestral and highly derived anatomical features has made morphoanatomical analyses of the xenarthran evolutionary history difficult, and previous molecular analyses failed to resolve the relationships within armadillo subfamilies. We investigated the presence/absence patterns of retroposons from approximately 7,400 genomic loci, identifying 35 phylogenetically informative elements and an additional 39 informative rare genomic changes (RGCs). DAS-short interspersed elements (SINEs), previously described only in the Dasypus novemcinctus genome, were found in all living armadillo genera, including the previously unsampled Chlamyphorus, but were noticeably absent in sloths. The presence/absence patterns of the phylogenetically informative retroposed elements and other RGCs were then compared with data from the DNA sequences of the more than 12-kb flanking regions of these retroposons. Together, these data provide the first fully resolved genus tree of xenarthrans. Interestingly, multiple evidence supports the grouping of Chaetophractus and Zaedyus as a sister group to Euphractus within Euphractinae, an association that was not previously demonstrated. Also, flanking sequence analyses favor a close phylogenetic relationship between Cabassous and Tolypeutes within Tolypeutinae. Finally, the phylogenetic position of the subfamily Chlamyphorinae is resolved by the noncoding sequence data set as the sister group of Tolypeutinae. The data provide a stable phylogenetic framework for further evolutionary investigations of xenarthrans and important information for defining conservation priorities to save the diversity of one of the most curious groups of mammals.

Background: Extant placental mammals are divided into four major clades (Laurasiatheria, Supraprimates, Xenarthra and Afrotheria). Given that Afrotheria is generally thought to root the eutherian tree in phylogenetic analysis of large nuclear gene data sets, the study of the organization of the genomes of afrotherian species provides new insights into the dynamics of mammalian chromosomal evolution. Here we test if there are chromosomal bands with a high tendency to break and reorganize in Afrotheria, and by analyzing the expression of aphidicolin-induced common fragile sites in three afrotherian species, whether these are coincidental with recognized evolutionary breakpoints., Results: We described 29 fragile sites in the aardvark (OAF) genome, 27 in the golden mole (CAS), and 35 in the elephant-shrew (EED) genome. We show that fragile sites are conserved among afrotherian species and these are correlated with evolutionary breakpoints when compared to the human (HSA) genome. Inddition, by computationally scanning the newly released opossum (Monodelphis domestica) and chicken sequence assemblies for use as outgroups to Placentalia, we validate the HSA 3/21/5 chromosomal synteny as a rare genomic change that defines the monophyly of this ancient African clade of mammals. On the other hand, support for HSA 1/19p, which is also thought to underpin Afrotheria, is currently ambiguous., Conclusion: We provide evidence that (i) the evolutionary breakpoints that characterise human syntenies detected in the basal Afrotheria correspond at the chromosomal band level with fragile sites, (ii) that HSA 3p/21 was in the amniote ancestor (i.e., common to turtles, lepidosaurs, crocodilians, birds and mammals) and was subsequently disrupted in the lineage leading to marsupials. Its expansion to include HSA 5 in Afrotheria is unique and (iii) that its fragmentation to HSA 3p/21 + HSA 5/21 in elephant and manatee was due to a fission within HSA 21 that is probably shared by all Paenungulata.

Recent rapid generation of genomic sequence data has allowed many researchers to perform comparative analyses in various mammalian species. However, characterization of transposable elements, such as short interspersed repetitive elements (SINEs), has not been reported for several mammalian groups. Because SINEs occupy a large portion of the mammalian genome, they are believed to have contributed to the constitution and diversification of the host genomes during evolution. In the present study, we characterized a novel SINE family in the anteater genomes and designated it the MyrSINE family. Typical SINEs consist of a tRNA-related, a tRNA-unrelated and an AT-rich (or poly-A) region. MyrSINEs have only tRNA-related and poly-A regions; they are included in a group called t-SINE. The tRNA-related regions of the MyrSINEs were found to be derived from tRNA(Gly). We demonstrate that the MyrSINE family can be classified into three subfamilies. Two of the MyrSINE subfamilies are distributed in the genomes of both giant anteater and tamandua, while the other is present only in the giant anteater. We discuss the evolutionary history of MyrSINEs and their relationship to the evolution of anteaters. We also speculate that the simple structure of t-SINEs may be a potential evolutionary source for the generation of the typical SINE structure.

The phylogenetic positions of the 4 clades, Euarchontoglires, Laurasiatheria, Afrotheria, and Xenarthra, have been major issues in the recent discussion of basal relationships among placental mammals. However, despite considerable efforts these relationships, crucial to the understanding of eutherian evolution and biogeography, have remained essentially unresolved. Euarchontoglires and Laurasiatheria are generally joined into a common clade (Boreoeutheria), whereas the position of Afrotheria and Xenarthra relative to Boreoeutheria has been equivocal in spite of the use of comprehensive amounts of nuclear encoded sequences or the application of genome-level characters such as retroposons. The probable reason for this uncertainty is that the divergences took place long time ago and within a narrow temporal window, leaving only short common branches. With the aim of further examining basal eutherian relationships, we have collected conserved protein-coding sequences from 11 placental mammals, a marsupial and a bird, whose nuclear genomes have been largely sequenced. The length of the alignment of homologous sequences representing each individual species is 2,168,859 nt. This number of sites, representing 2840 protein-coding genes, exceeds by a considerable margin that of any previous study. The phylogenetic analysis joined Xenarthra and Afrotheria on a common branch, Atlantogenata. This topology was found to fit the data significantly better than the alternative trees.

Molecular studies have led recently to the proposal of a new super-ordinal arrangement of the 18 extant Eutherian orders. From the four proposed super-orders, Afrotheria and Xenarthra were considered the most basal. Chromosome-painting studies with human probes in these two mammalian groups are thus key in the quest to establish the ancestral Eutherian karyotype. Although a reasonable amount of chromosome-painting data with human probes have already been obtained for Afrotheria, no Xenarthra species has been thoroughly analyzed with this approach. We hybridized human chromosome probes to metaphases of species (Dasypus novemcinctus, Tamandua tetradactyla, and Choloepus hoffmanii) representing three of the four Xenarthra families. Our data allowed us to review the current hypotheses for the ancestral Eutherian karyotype, which range from 2n = 44 to 2n = 48. One of the species studied, the two-toed sloth C. hoffmanii (2n = 50), showed a chromosome complement strikingly similar to the proposed 2n = 48 ancestral Eutherian karyotype, strongly reinforcing it., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

To better understand the evolution of genome organization of eutherian mammals, comparative maps based on chromosome painting have been constructed between human and representative species of three eutherian orders: Xenarthra, Pholidota, and Eulipotyphla, as well as between representative species of the Carnivora and Pholidota. These maps demonstrate the conservation of such syntenic segment associations as HSA3/21, 4/8, 7/16, 12/22, 14/15 and 16/19 in Eulipotyphla, Pholidota and Xenarthra and thus further consolidate the notion that they form part of the ancestral karyotype of the eutherian mammals. Our study has revealed many potential ancestral syntenic associations of human chromosomal segments that serve to link the families as well as orders within the major superordinial eutherian clades defined by molecular markers. The HSA2/8 and 7/10 associations could be the cytogenetic signatures that unite the Xenarthrans, while the HSA1/19p could be a putative signature that links the Afrotheria and Xenarthra. But caution is required in the interpretation of apparently shared syntenic associations as detailed analyses also show examples of apparent convergent evolution that differ in breakpoints and extent of the involved segments.

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.

Recent comprehensive studies of DNA sequences support the monophyly of Afrotheria, comprising elephants, sirenians (dugongs and manatees), hyraxes, tenrecs, golden moles, aardvarks, and elephant shrews, as well as that of Paenungulata, comprising elephants, sirenians, and hyraxes. However, phylogenetic relationships among paenungulates, as well as among nonpaenungulates, have remained ambiguous. Here we applied an extensive retroposon analysis to these problems to support the monophyly of aardvarks, tenrecs, and golden moles, with elephant shrews as their sister group. Regarding phylogenetic relationships in Paenungulata, we could characterize only one informative locus, although we could isolate many insertions specific to each of three lineages, namely, Proboscidea, Sirenia, and Hyracoidea. These data prompted us to reexamine phylogenetic relationships among Paenungulata using 19 nuclear gene sequences resulting in three different analyses, namely, short interspersed element (SINE) insertions, nuclear sequence analyses, and morphological cladistics, supporting different respective phylogenies. We concluded that these three lineages diverged very rapidly in a very short evolutionary period, with the consequence that ancestral polymorphism present in the last common ancestor of Paenungulata results in such incongruence. Our results suggest the rapid fixation of many large-scale morphological synapomorphies for Tethytheria; implications of this in relation to the morphological evolution in Paenungulata are discussed.

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.

Comparative cytogenetic studies on Xenarthra, one of the most basal mammalian clades in the Placentalia, are virtually absent, being restricted largely to descriptions of conventional karyotypes and diploid numbers. We present a molecular cytogenetic comparison of chromosomes from the two-toed (Choloepus didactylus, 2n = 65) and three-toed sloth species (Bradypus tridactylus, 2n = 52), an anteater (Tamandua tetradactyla, 2n = 54) which, together with some data on the six-banded armadillo (Euphractus sexcinctus, 2n = 58), collectively represent all the major xenarthran lineages. Our results, based on interspecific chromosome painting using flow-sorted two-toed sloth chromosomes as painting probes, show the sloth species to be karyotypically closely related but markedly different from the anteater. We also test the synteny disruptions and segmental associations identified within Pilosa (anteaters and sloths) against the chromosomes of the six-banded armadillo as outgroup taxon. We could thus polarize the 35 non-ambiguously identified chromosomal changes characterizing the evolution of the anteater and sloth genomes and map these to a published sequence-based phylogeny for the group. These data suggest a low rate of genomic repatterning when placed in the context of divergence estimates based on molecular and fossil data. Finally, our results provide a glimpse of a likely ancestral karyotype for the extant Xenarthra, a pivotal group for understanding eutherian genome evolution.

Long interspersed nuclear elements (LINEs) comprise about 21% of the human genome (of which L1 is most abundant) and are preferentially accumulated in AT-rich regions, as well as the X and Y chromosomes. Most knowledge of L1 distribution in mammals is restricted to human and mouse. Here we report the first investigation of L1 distribution in the genomes of a wide variety of eutherian mammals, including species in the two basal clades, Afrotheria and Xenarthra. Our results show L1 accumulation on the X of all eutherian mammals, an observation consistent with an ancestral involvement of these elements in the X-inactivation process (the Lyon repeat hypothesis). Surprisingly, conspicuous accumulation of L1 in AT-rich regions of the genome was not observed in any species outside of Euarchontoglires (represented by human, mouse and rabbit). Although several features were common to most species investigated, our comprehensive survey shows that the patterns observed in human and mouse are, in many aspects, far from typical for all mammals. We discuss these findings with reference to models that have previously been proposed to explain the AT distribution bias of L1 in human and mouse, and how this relates to the evolution of these elements in other eutherian genomes.

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.

The Afrotheria, a supraordinal grouping of mammals whose radiation is rooted in Africa, is strongly supported by DNA sequence data but not by their disparate anatomical features. We have used flow-sorted human, aardvark, and African elephant chromosome painting probes and applied reciprocal painting schemes to representatives of two of the Afrotherian orders, the Tubulidentata (aardvark) and Proboscidea (elephants), in an attempt to shed additional light on the evolutionary affinities of this enigmatic group of mammals. Although we have not yet found any unique cytogenetic signatures that support the monophyly of the Afrotheria, embedded within the aardvark genome we find the strongest evidence yet of a mammalian ancestral karyotype comprising 2n = 44. This karyotype includes nine chromosomes that show complete conserved synteny to those of man, six that show conservation as single chromosome arms or blocks in the human karyotype but that occur on two different chromosomes in the ancestor, and seven neighbor-joining combinations (i.e., the synteny is maintained in the majority of species of the orders studied so far, but which corresponds to two chromosomes in humans). The comparative chromosome maps presented between human and these Afrotherian species provide further insight into mammalian genome organization and comparative genomic data for the Afrotheria, one of the four major evolutionary clades postulated for the Eutheria.

Extant xenarthrans (armadillos, anteaters and sloths) are among the most derived placental mammals ever evolved. South America was the cradle of their evolutionary history. During the Tertiary, xenarthrans experienced an extraordinary radiation, whereas South America remained isolated from other continents. The 13 living genera are relics of this earlier diversification and represent one of the four major clades of placental mammals. Sequences of the three independent protein-coding nuclear markers alpha2B adrenergic receptor (ADRA2B), breast cancer susceptibility (BRCA1), and von Willebrand Factor (VWF) were determined for 12 of the 13 living xenarthran genera. Comparative evolutionary dynamics of these nuclear exons using a likelihood framework revealed contrasting patterns of molecular evolution. All codon positions of BRCA1 were shown to evolve in a strikingly similar manner, and third codon positions appeared less saturated within placentals than those of ADRA2B and VWF. Maximum likelihood and Bayesian phylogenetic analyses of a 47 placental taxa data set rooted by three marsupial outgroups resolved the phylogeny of Xenarthra with some evidence for two radiation events in armadillos and provided a strongly supported picture of placental interordinal relationships. This topology was fully compatible with recent studies, dividing placentals into the Southern Hemisphere clades Afrotheria and Xenarthra and a monophyletic Northern Hemisphere clade (Boreoeutheria) composed of Laurasiatheria and Euarchontoglires. Partitioned likelihood statistical tests of the position of the root, under different character partition schemes, identified three almost equally likely hypotheses for early placental divergences: a basal Afrotheria, an Afrotheria + Xenarthra clade, or a basal Xenarthra (Epitheria hypothesis). We took advantage of the extensive sampling realized within Xenarthra to assess its impact on the location of the root on the placental tree. By resampling taxa within Xenarthra, the conservative Shimodaira-Hasegawa likelihood-based test of alternative topologies was shown to be sensitive to both character and taxon sampling.

Recent analyses of nucleotide sequence data suggest that living placental mammals belong to one of four superorders. The early divergence of these groups was followed by long periods of geographical isolation, due to the break up of continental land masses, allowing for convergent evolution of similar traits in different superorders. As an example, the transition from epitheliochorial to haemochorial placentation occurred independently in bats, rodents, anthropoid primates, armadillos and others. A group of ancient African mammals is suggested by the molecular data, but is not fully supported by morphological evidence. The hypothesis is, however, consistent with some of the data on fetal membranes, suggesting that it would be worthwhile to study the early development of tenrecs, golden moles and elephant shrews. Analyses of fetal membrane traits that group the tarsiers with anthropoid primates, and separate them from the lemurs, are challenged by the molecular data. Other relatives of the primates seem to include tree shrews and flying lemurs, and little is known about the fetal membranes of the latter group. Comparative studies of placental function normally are confined to primates, rodents, lagomorphs and domestic animals: the biological diversity represented by mammals that evolved in ancient Africa and South America is not represented. Therefore, future comparative studies should strive to include species such as the rock hyrax and the armadillo., (Copyright 2001 Harcourt Publishers Ltd.)

Xenarthra (Edentata) is an extremely diverse mammalian order whose modern representatives are the armadillos, anteaters, and sloths. The phylogeny of these groups is poorly resolved. This is particularly true for the sloths (phyllophagans), originally a large and diverse group now reduced to two genera in two different families. Both morphological analyses and molecular analyses of rDNA genes of living and extinct sloths have been used with limited success to elucidate their phylogeny. In an attempt to clarify relationships among the sloths, DNA was extracted and mitochondrial cytochrome b gene sequences were determined from representatives of two extinct groups of sloths (Mylodontidae and Megatheriidae), their two living relatives (two-toed sloths [Megalonychidae], three-toed sloths [Bradypodidae]), anteaters and armadillos. A consistent feature of the latter two species was the nuclear copies of cytochrome b gene sequences. Several methods of phylogenetic reconstruction were applied to the sequences determined, and the results were compared with 12S rDNA sequences obtained in previous studies. The cytochrome b gene exhibited a phylogenetic resolving power similar to that of the 12S rDNA sequences. When both data sets were combined, they tended to support the grouping of two-toed sloths with mylodontids and three-toed sloths with megatheriids. The results strengthen the view that the two families of living sloths adapted independently to an arboreal life-style., (Copyright 2000 Academic Press.)

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.

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein alpha A-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, or in 2 species of marsupials, or in 34 nonmammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the alpha A-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 76% to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the estimates obtained, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than do the two trees that assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is > 0.99. Thus, this deletion in alpha A-crystallin gives strong molecular support for the monophyly of this old and diverse order.

An outstanding problem in mammal phylogeny is the relationship of the aardvark (Orycteropus afer), the only living species of the order Tubulidentata, to the extant eutherian lineages. In order to examine this problem the complete mitochondrial DNA (mtDNA) molecule of the aardvark was sequenced and analysed. The aardvark tRNA-Ser (UCN) differs from that of other mammalian mtDNAs reported and appears to have reversed to the ancestral secondary structure of non-mammalian vertebrates and mitochondrial tRNAs in general. Phylogenetic analysis of 12 concatenated protein-coding genes (3325 amino acids) included the aardvark and 15 additional eutherians, two marsupials and a monotreme. The most strongly supported tree identified the aardvark as a sister group of a clade including the armadillo (Xenarthra) and the Cetferungulata (carnivores, perissodactyls, artiodactyls and cetaceans). By applying three molecular calibration points the divergence between the aardvark and armadillo-cetferungulates was estimated at ca. 90 million years before present.

A solution to higher level mammalian phylogeny is going to depend on the congruent establishment of superordinal groupings followed by a linking together of these clades. We present congruent and convincing evidence from four disparate nuclear protein coding genes and from a tandem alignment of the 12S-16S mitochondrial region, for a superordinal clade of endemic African mammals that includes elephant shrews, aardvarks, golden mole, elephants, sirenians, and hyraxes. Because of strong support for golden mole as part of this clade, the Insectivora are rendered paraphyletic or polyphyletic, with constrained monophyly of the insectivores judged significantly worse in the vast majority of tests. Branching arrangement within this clade remains highly uncertain; however, a tandem alignment of the protein coding genes suggests elephant shrew is the earliest African lineage. None of the individual data sets or combinations of data sets support the widely held view of a mirorder Tethytheria (Sirenia/Proboscidea), although only a tandem alignment of protein coding and mitochondrial loci significantly rejects this association. The majority of the data sets and analyses provide strong support for Caviomorpha as part of a monophyletic Rodentia., (Copyright 1998 Academic Press.)

Phylogenetic relationships among 27 extant mammalian species (representing 15 placental orders) were studied using sequences of exon 28 of the gene encoding von Willebrand Factor (vWF), a glycoprotein which functions in blood clotting. Analysis of sequences coding for vWF revealed evidence for several subordinal and superordinal groupings, but the earliest branching sequence of placental mammals was left largely unresolved. Strong support was found for a monophyletic clade consisting of elephants, sea cows, hyraxes, aardvarks, and elephant shrews. This systematic placement of the elephant shrews agrees strongly with two other molecular data sets (interphotoreceptor retinoid binding protein and alpha-lens crystallins) and is consistent with analysis of fossil elephant shrews recently discovered in north Africa. Evidence from vWF sequences agrees with a number of previous molecular and morphological studies in providing strong support for the monophyly of both bats and rodents. The orders Primates, Proboscidea, Carnivora, Perissodactyla, and Artiodactyla were represented by more than one species which joined in each case to form a monophyletic order.

We examined protein polymorphism of Chinese pangolins (Manis pentadactyla) from Yunnan Province of China, including two forms of three brown and nine dusky Chinese pangolins. Sixty-two genetic loci were screened; 12 loci were found to be polymorphic. The percentage of polymorphic loci (P) is 0.194, the mean individual heterozygosity (H) is 0.078, and the mean number of alleles (A) is 1.258. Furthermore, we calculated the genetic distance (D) between the two forms and found a low level of genetic divergence (D = 0.0206) between them, which indicates an almost-indistinguishable divergence at the level of proteins.

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51-56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.

The somatic and meiotic chromosomes of twenty specimens of the Dasypodidae were studied. The wild specimens belonged to the genera Cabassous, Euphractus and Dasypus. The karyotype of D. novemcinctus showed a diploid chromosome number of 64, although some variation was observed in one specimen where 2n was 65, due to a supernumerary chromosome, and another had a dimorphic pair. D. septemcinctus also had a diploid chromosome number of 64, and showed two variant types due to pericentric inversions and/or centric fusions. E. sexcinctus and C. unicinctus had diploid chromosome numbers of 58 and 50, respectively. These species did not show variant chromosome types, and this was confirmed in meiotic studies. Analysis of the meiotic epithelium of species of Dasypus including the analysis of the synaptonemal complex in pachytene nuclei, showed similar findings to those commonly reported in mammalian meiosis, especially in relation to pairing and formation of the sex bivalent. The evolutionary implications of the chromosome changes in relation to organic evolution are discussed.

C- and G-banding patterns of Orycteropus afer are described on the basis of fibroblast cultures obtained from a female individual.

Skeletal muscle myoglobin of the aardvark (Orycteropus afer) was isolated and its primary structure determined. The amino-acid sequence was then used in conjunction with previously established myoglobin sequences to evaluate the phylogenetic relationships of the aardvark. The most parsimonious trees constructed from this myoglobin sequence data either alone or when combined with lens alpha-crystallin A sequence data depict the aardvark lineage as one of the most ancient among Eutheria.