Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

The nine-banded armadillo (Dasypus novemcinctus) is the most widespread xenarthran species across the Americas. Recent studies have suggested it is composed of four morphologically and genetically distinct lineages of uncertain taxonomic status. To address this issue, we used a museomic approach to sequence 80 complete mitogenomes and capture 997 nuclear loci for 71 Dasypus individuals sampled across the entire distribution. We carefully cleaned up potential genotyping errors and cross contaminations that could blur species boundaries by mimicking gene flow. Our results unambiguously support four distinct lineages within the D. novemcinctus complex. We found cases of mito-nuclear phylogenetic discordance but only limited contemporary gene flow confined to the margins of the lineage distributions. All available evidence including the restricted gene flow, phylogenetic reconstructions based on both mitogenomes and nuclear loci, and phylogenetic delimitation methods consistently supported the four lineages within D. novemcinctus as four distinct species. Comparable genetic differentiation values to other recognized Dasypus species further reinforced their status as valid species. Considering congruent morphological results from previous studies, we provide an integrative taxonomic view to recognise four species within the D. novemcinctus complex: D. novemcinctus, D. fenestratus, D. mexicanus, and D. guianensis sp. nov., a new species endemic of the Guiana Shield that we describe here. The two available individuals of D. mazzai and D. sabanicola were consistently nested within D. novemcinctus lineage and their status remains to be assessed. The present work offers a case study illustrating the power of museomics to reveal cryptic species diversity within a widely distributed and emblematic species of mammals.

Comparative metal analysis in a species assemblage of mammals from the Southeastern United States.

Metal pollution commonly occurs in many terrestrial environments and may pose a threat for the animals inhabiting such areas. Here, we present concentrations of six metals (cadmium [Cd], copper [Cu], nickel [Ni], lead [Pb], selenium [Se], and zinc [Zn]) in the liver tissues of seven species of mammals obtained from a study that examined the impact of mesopredator removal on northern bobwhite (Colinus virginianus) populations. A total of 1326 samples were collected from 2003 to 2006 at four sites in southwest Georgia and north Florida from nine-banded armadillos (Dasypus novemcinctus), bobcats (Lynx rufus), feral cats (Felis catus), coyotes (Canis latrans), grey foxes (Urocyon cinereoargenteus), opossums (Didelphis virginiana), and raccoons (Procyon lotor). Data from armadillos, bobcats, opossums, and raccoons were published previously to examine age, sex, spatial (between sites), and temporal (between years) variation. In this paper, we present similar comparisons for the remaining three species as well as comparisons of metal concentrations among all seven species. Concentrations of Cu and Pb exhibited strong negative relationships with body weight in coyotes, while Ni was positively correlated with weight in feral cats. Concentrations of these metals, as well as the other two tested (Cd and Zn), were not significantly correlated with one another in any of the three species. The only sex difference in liver metal concentrations was observed in female feral cats, which had higher levels of Pb than did males. Coyotes exhibited significant differences in Cu concentrations between sites and between years (2005 versus 2006). We also found significant differences between sites in Pb concentrations for both feral cats and grey foxes. There were significant differences in metal concentrations among all seven species for all metals except Cd. With the exception of Cd and Se (tested only in bobcats and opossums), a three-way ANOVA with species, year, and site as the three factors revealed significant differences among species for every metal but only a single main effect of year for Cu, and no main effects of site. In sum, our results provide an extensive survey of metal concentrations in a diverse assemblage of mammals and suggest that metal accumulation may be heavily influenced by species identity, which in turn may reflect ecological lifestyle.

Vertebral bending mechanics and xenarthrous morphology in the nine-banded armadillo (Dasypus novemcinctus).

The vertebral column has evolved to accommodate the broad range of locomotor pressures found across vertebrate lineages. Xenarthran (armadillos, sloths and anteaters) vertebral columns are characterized by xenarthrous articulations, novel intervertebral articulations located in the posterior trunk that are hypothesized to stiffen the vertebral column to facilitate digging. To determine the degree to which xenarthrous articulations impact vertebral movement, we passively measured compliance and range of motion during ventroflexion, dorsiflexion and lateral bending across the thoracolumbar region of the nine-banded armadillo, Dasypus novemcinctus Patterns of bending were compared with changes in vertebral morphology along the column to determine which morphological features best predict intervertebral joint mechanics. We found that compliance was lower in post-diaphragmatic, xenarthrous vertebrae relative to pre-xenarthrous vertebrae in both sagittal and lateral planes of bending. However, we also found that range of motion was higher in this region. These changes in mechanics are correlated with the transition from pre-xenarthrous to xenarthrous vertebrae, as well as with the transition from thoracic to lumbar vertebrae. Our results thus substantiate the hypothesis that xenarthrous articulations stiffen the vertebral column. Additionally, our data suggest that xenarthrous articulations, and their associated enlarged metapophyses, also act to increase the range of motion of the post-diaphragmatic region. We propose that xenarthrous articulations perform the dual role of stiffening the vertebral column and increasing mobility, resulting in passively stable vertebrae that are capable of substantial bending under appropriate loads.

Metal accumulation in wild-caught opossum.

The Virginia opossum (Didelphis virginiana) is widespread in the USA, ranging south through Latin America. The ecology of opossums is such that they are in frequent contact with soils, suggesting that they may function as a valuable bioindicator for chemical contamination in terrestrial environments. Surprisingly, there have been virtually no toxicology studies on opossums. Here, we provide the first analysis of metal contaminants in opossum liver tissues. Liver samples were obtained from 471 opossums, collected from 2003 to 2006, at four sites in North Florida and South Georgia, USA, and concentrations of copper, lead, nickel, selenium, and zinc were measured. We found little evidence of age differences in the concentration of any of the metals. However, there were at least some significant differences between years, males and females, and between sites for each metal, although the pattern of these differences was not always consistent across metals. Concentrations of metals in liver tissue were positively correlated with one another, primarily of each metal (except Pb) with zinc. Reference levels of metal contaminants are not available for opossums, but concentrations of Cu, Ni, Pb, and Zn in our samples were for the most part significantly higher than those reported from liver tissues of nine-banded armadillos (Dasypus novemcinctus) collected at the same sites and in the same years. Data from other small mammals studied elsewhere further indicate that metal concentrations in opossums were high, but at this time, it is not possible to determine if these elevated levels generated toxicity. The substantial temporal and spatial variation we found in metal concentrations suggests that determination of baseline levels for opossums may not be straightforward. Nonetheless, this is the first study quantifying metal accumulation in the livers of Didelphis virginiana and, as such, provides an important starting point for future research.

Metal accumulation in bobcats in the Southeastern USA.

Bobcats (Lynx rufus) are wide-ranging mammals found throughout the continental USA. As carnivores near the top of their food chain, bobcats would seem to be a useful bioindicator of metal pollution in terrestrial environments. However, there is very limited research on bobcats in toxicology studies. Here, we offer the first analysis of metal (copper, selenium, silver, and zinc) contaminants in the livers of wild bobcats. Liver tissues from 120 adult bobcats (i.e., estimated to be ≥1 year old) were collected from 2003 to 2006 at four sites in Georgia and Florida, USA that experienced relatively similar levels of human disturbance. We found no differences in metal concentrations between males and females. At two of the sites sampled over three consecutive years, there was substantial year-to-year variation in the concentrations of Cu, Se, and Zn. We also documented some variation between sites, but only between sites sampled in different years, which may reflect additional temporal, rather than spatial, variation. Concentrations of Cu and Ag were significantly positively correlated with one another, as were concentrations of Se and Zn. Contrary to expectation, there were no significant relationships between body weight and metal concentrations. Finally, comparison with results from previous metal toxicology studies of nine-banded armadillos (Dasypus novemcinctus) and Virginia opossums (Didelphis virgianus), collected from the same sites during the same years, showed differential patterns of accumulation across species, suggesting that ecological lifestyle is an important influence on metal accumulation. This study provides reference levels of metal contaminants in the liver of bobcats as well as insight into metal accumulation in a top level carnivore.

Zoonotic Leprosy in the Southeastern United States.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae-infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.

Seroprevalence of Leprosy in Nine-Banded Armadillos (Dasypus novemcinctus) from Tennessee, USA, 2021-22.

The nine-banded armadillo (Dasypus novemcinctus) is currently considered an invasive species in parts of its range in the USA, and this range continues to expand to the north and east. Nine-banded armadillos are one of a handful of mammals known to contract leprosy (also known as Hansen's disease); range expansion thus leads to public health concerns about whether this might increase human exposure to infected animals. We collected blood samples from 61 road-killed armadillos over two summers (2021 and 2022) in Tennessee, a US state near the northern extreme of the species' current range, and screened them for exposure to Mycobacterium leprae, the causative agent of leprosy. All animals were seronegative, providing no evidence that range expansion is increasing the distribution of leprosy in the US.

Metal accumulation in wild nine-banded armadillos.

Nine-banded armadillos (Dasypus novemcinctus) are widespread and abundant New World mammals with a lifestyle that entails prolonged, intimate contact with soils. Thus, armadillos would seem a promising candidate as a sentinel species to monitor chemical contamination in terrestrial ecosystems. Surprisingly, there have been virtually no toxicology studies on armadillos. Here, we provide the first analysis of metal contaminants for wild armadillos. Liver tissues were obtained from 302 armadillos collected at 6 sites in Georgia and Florida, USA that varied in their extent of human disturbance, from rural pine plantations to highly modified military/space installations. Data were stratified by age (juvenile and adult), sex, and site. Temporal (yearly) variation was examined at two of the sites that were sampled over three consecutive years. Concentrations of aluminum, cadmium, copper, nickel, lead, and zinc were measured in liver samples from each site. Although reference levels are not available for armadillos, accumulated metal concentrations were comparable to those reported for other mammals. We found no evidence of sex or age differences in the concentrations of any metal, except for Cd (age) and Pb (sex and age). However, concentrations of most metals varied substantially across sites and over time. Finally, concentrations of many metals were positively correlated with one another, suggesting that they likely co-occurred in some areas. Collectively, this study indicates the utility of armadillos as a sentinel species for studies of metal contamination in terrestrial systems, and highlights the need for further studies of other toxicants in these animals.

Metagenomics uncovers dietary adaptations for chitin digestion in the gut microbiota of convergent myrmecophagous mammals.

IMPORTANCE: Myrmecophagous mammals are specialized in the consumption of ants and/or termites. They do not share a direct common ancestor and evolved convergently in five distinct placental orders raising questions about the underlying adaptive mechanisms involved and the relative contribution of natural selection and phylogenetic constraints. Understanding how these species digest their prey can help answer these questions. More specifically, the role of their gut microbial symbionts in the digestion of the insect chitinous exoskeleton has not been investigated in all myrmecophagous orders. We generated 29 new gut metagenomes from nine myrmecophagous species to reconstruct more than 300 bacterial genomes in which we identified chitin-degrading enzymes. Studying the distribution of these chitinolytic bacteria among hosts revealed both shared and specific bacteria between ant-eating species. Overall, our results highlight the potential role of gut symbionts in the convergent dietary adaptation of myrmecophagous mammals and the evolutionary mechanisms shaping their gut microbiota.

Is leprosy spreading among nine-banded armadillos in the southeastern United States?

In the United States, nine-banded armadillo (Dasypus novemcinctus) populations are derived from two sources: (1) a continuous range expansion from Mexico led to western populations, some of which, particularly along the western Gulf Coast and west side of the Mississippi River delta, exhibit persistently high rates of leprosy infection, and (2) a small group of animals released from captivity in Florida gave rise to eastern populations that were all considered leprosy free. Given that western and eastern populations have now merged, an important question becomes, to what extent is leprosy spreading into formerly uninfected populations? To answer this question, we sampled 500 animals from populations in Mississippi, Alabama, and Georgia. Analyses of nuclear microsatellite DNA markers confirmed the historic link between source populations from Texas and Florida, but did not permit resolution of the extent to which these intermediate populations represented eastern versus western gene pools. Prevalence of leprosy was determined by screening blood samples for the presence of antibodies against Mycobacterium leprae and via polymerase chain reaction amplification of armadillo tissues to detect M. leprae DNA. The proportion of infected individuals within each population varied from 0% to 10%. Although rare, a number of positive individuals were identified in eastern sites previously considered uninfected. This indicates leprosy may be spreading eastward and calls into question hypotheses proposing leprosy infection is confined because of ecologic constraints to areas west of the Mississippi River.

PATTERNS OF MYCOBACTERIUM LEPRAE INFECTION IN WILD NINE-BANDED ARMADILLOS (DASYPUS NOVEMCINCTUS) IN MISSISSIPPI, USA.

The nine-banded armadillo ( Dasypus novemcinctus ) is the only known nonhuman reservoir of Mycobacterium leprae , the causative agent of Hansen's disease or leprosy. We conducted a 6-yr study on a wild population of armadillos in western Mississippi that was exposed to M. leprae to evaluate the importance of demographic and spatial risk factors on individual antibody status. We found that spatially derived covariates were not predictive of antibody status. Furthermore, analyses revealed no evidence of clustering by antibody-positive individuals. Lactating females and adult males had higher odds of being antibody positive than did nonlactating females. No juveniles or yearlings were antibody positive. Results of these analyses support the hypothesis that M. leprae infection patterns are spatially homogeneous within this armadillo population. Further research related to movement patterns, contact among individuals, antibody status, and environmental factors could help address hypotheses related to the role of environmental transmission on M. leprae infection and the mechanisms underlying the differential infection patterns among demographic groups.

Molecular documentation of polyembryony and the micro-spatial dispersion of clonal sibships in the nine-banded armadillo, Dasypus novemcinctus.

A battery of allelic markers at highly polymorphic microsatellite loci was developed and employed to confirm genetically, the clonal nature of sibships in nine-banded armadillos. This phenomenon of consistent polyembryony, otherwise nearly unknown among the vertebrates, was capitalized upon to describe the micro-spatial distributions of numerous clonal sibships in a natural population of armadillos. Adult clone mates were significantly more dispersed than were juvenile sibs, suggesting limited opportunities for altruistic behavioural interactions among mature individuals. These results, and considerations of armadillo natural history, suggest that evolutionary explanations for polyembryony in this species may not reside in the kinds of ecological and kin selection theories relevant to some of the polyembryonic invertebrates. Rather, polyembryony in armadillos may be associated evolutionarily with other reproductive peculiarities of the species, including delayed uterine implantation of a single egg.

Genetic maternity and paternity in a local population of armadillos assessed by microsatellite DNA markers and field data.

Genetic data from polymorphic microsatellite loci were employed to estimate paternity and maternity in a local population of nine-banded armadillos (Dasypus novemcinctus) in northern Florida. The parentage assessments took advantage of maximum likelihood procedures developed expressly for situations when individuals of neither gender can be excluded a priori as candidate parents. The molecular data for 290 individuals, interpreted alone and in conjunction with detailed biological and spatial information for the population, demonstrate high exclusion probabilities and reasonably strong likelihoods of genetic parentage assignment in many cases; low mean probabilities of successful reproductive contribution to the local population by individual armadillo adults in a given year; and statistically significant microspatial associations of parents and their offspring. Results suggest that molecular assays of highly polymorphic genetic systems can add considerable power to assessments of biological parentage in natural populations even when neither parent is otherwise known.