A molecular phylogeny of two extinct sloths

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.     

A syndrome of mutualism reinforces the lifestyle of a sloth

Arboreal herbivory is rare among mammals. The few species with this lifestyle possess unique adaptions to overcome size-related constraints on nutritional energetics. Sloths are folivores that spend most of their time resting or eating in the forest canopy. A three-toed sloth will, however, descend its tree weekly to defecate, which is risky, energetically costly and, until now, inexplicable. We hypothesized that this behaviour sustains an ecosystem in the fur of sloths, which confers cryptic nutritional benefits to sloths. We found that the more specialized three-toed sloths harboured more phoretic moths, greater concentrations of inorganic nitrogen and higher algal biomass than the generalist two-toed sloths. Moth density was positively related to inorganic nitrogen concentration and algal biomass in the fur. We discovered that sloths consumed algae from their fur, which was highly digestible and lipid-rich. By descending a tree to defecate, sloths transport moths to their oviposition sites in sloth dung, which facilitates moth colonization of sloth fur. Moths are portals for nutrients, increasing nitrogen levels in sloth fur, which fuels algal growth. Sloths consume these algae-gardens, presumably to augment their limited diet. These linked mutualisms between moths, sloths and algae appear to aid the sloth in overcoming a highly constrained lifestyle.     

Hematological and serum chemistry profiles of free-ranging southern two-toed sloths in French Guiana.

Free-ranging southern two-toed sloths (Choloepus didactylus) were translocated during the flooding of a forest at a hydroelectric dam site in French Guiana. Over an 11 mo period blood samples were collected from 90 sloths (38 males, 52 females) in order to determine hematological and serum chemistry reference values. Mean values and range of values were calculated for 13 hematological and 21 serum chemistry parameters. Variations associated with sex, age and reproductive status were identified. Males had a significantly lower red blood cell count than females. Immature animals had more monocytes while adults had more neutrophils and higher mean corpuscular hemoglobin concentration. Aspartate aminotransferase and triglyceride values were higher in young than in adult sloths but uric acid was lower. Lactating females showed lower red blood cells count and iron levels than non-lactating females. These profiles will help to provide reliable baseline data for medical evaluation of sloths.     

A high resolution map of mammalian X chromosome fragile regions assessed by large-scale comparative genomics

Chromosomal evolution involves multiple changes at structural and numerical levels. These changes, which are related to the variation of the gene number and their location, can be tracked by the identification of syntenic blocks (SB). First reports proposed that ~180–280 SB might be shared by mouse and human species. More recently, further studies including additional genomes have identified up to ~1,400 SB during the evolution of eutherian species. A considerable number of studies regarding the X chromosome’s structure and evolution have been undertaken because of its extraordinary biological impact on reproductive fitness and speciation. Some have identified evolutionary breakpoint regions and fragile sites at specific locations in the human X chromosome. However, mapping these regions to date has involved using low-to-moderate resolution techniques. Such scenario might be related to underestimating their total number and giving an inaccurate location. The present study included using a combination of bioinformatics methods for identifying, at base-pair level, chromosomal rearrangements occurring during X chromosome evolution in 13 mammalian species. A comparative technique using four different algorithms was used for optimizing the detection of hotspot regions in the human X chromosome. We identified a significant interspecific variation in SB size which was related to genetic information gain regarding the human X chromosome. We found that human hotspot regions were enriched by LINE-1 and Alu transposable elements, which may have led to intraspecific chromosome rearrangement events. New fragile regions located in the human X chromosome have also been postulated. We estimate that the high resolution map of X chromosome fragile sites presented here constitutes useful data concerning future studies on mammalian evolution and human disease.     

Premaxillae of the Extinct Megalonychid Sloths <Emphasis Type="Italic">Acratocnus</Emphasis>, <Emphasis Type="Italic">Neocnus</Emphasis>, and <Emphasis Type="Italic">Megalonyx</Emphasis>, and their Phylogenetic Implications (Mammalia,Xenarthra)

In most folivorans, the premaxilla is loosely attached to the maxilla, so that it is often missing in otherwise very well-preserved fossil skulls. Despite its infrequent preservation in sloths, the premaxilla has been shown to have phylogenetically significant variation among the taxa that do preserve the element. In the family Megalonychidae, the premaxilla is known only in the early taxon Eucholoeops (Santacrucian South American Land Mammal Age [SALMA]), the extant two-toed sloth Choloepus, and the North American Neogene taxon Megalonyx, the last described only in an unpublished Master’s thesis. We report here the discovery of the premaxilla in two genera of extinct megalonychids, Neocnus and Acratocnus. These small bodied, semiarboreal megalonychid sloths are endemic to the islands of the Greater Antilles. Though the presence of sloths in the Caribbean dates at least to the late Oligocene, the best known taxa derive from Pleistocene to Holocene cave deposits in Puerto Rico, Hispaniola, and Cuba. We also describe the premaxilla in two species of Megalonyx from North America, the Blancan North American Land Mammal Age (NALMA) M. leptostomus and Rancholabrean NALMA M. jeffersonii. These species show a progressive reorientation of the premaxilla within Megalonyx from a primitive horizontal element to a nearly vertical element, and some significant changes in the anatomy of the incisive foramen. Morphological evidence suggests that a broadened, plate-like premaxilla constitutes a synapomorphy for the entire clade Megalonychidae. Furthermore, although Eucholoeops retains a short anterior process of the premaxilla like that of megatherioid sloths, this process is lacking in the other megalonychids, suggesting that the loss of this process may unite late Miocene to Recent megalonychids.     

Erratum

Feet of two-toed sloths (Choloepus) are long, narrow, hook-like appendages with only three functional digits, numbers II, III, and IV; Rays I and V are represented by metatarsals. Proximal phalanges of complete digits are little more than proximal and distal articulating surfaces. All interphalangeal joints are restricted, by interlocking surfaces, to flexion and extension. Ankle and transverse tarsal joints, however, allow extreme flexion and inversion of foot. Powerful digital flexion is augmented by several muscles from extensor compartment of leg. Intrinsic foot musculature is reduced to flexors and extensors but these, with the exception of lumbricals, are large and well developed. Choloepus uses its feet much like hooks with distal phalanges and covering claws forming the “hook” element. These hook-like appendages are seemingly best suited for supports less than 50 mm in diameter suggesting that two-toed sloths may prefer supports of this size in their natural habitat.     

The Convergent Evolution of Suspensory Posture and Locomotion in Tree Sloths

Recent phylogenetic analyses imply a distant relationship and long separated evolution of two-toed sloths (Choloepus) and three-toed sloths (Bradypus). No known fossil sloth is interpreted to have been suspensory. As a consequence, the suspensory posture and locomotion of the extant genera likely evolved convergently in both lineages, forming a new framework for the analysis of functional aspects of the locomotor apparatus of extant tree sloths. The suspensory posture and locomotion has altered functional demands from the phylogenetically plesiomorphic non-suspensory pronograde situation. Here, anatomical traits that have been argued to be of adaptive significance for quadrupedal suspensory locomotion are reviewed and the evolution of these traits is discussed in light of the new framework. Experimental data are largely limited to Choloepus, but help to deduce functional aspects of the anatomy in Bradypus as well. The most important adaptive traits are hands and feet modified into relatively rigid hook-like appendages, great mobility of all joints proximal to the midcarpal and transverse tarsal joints, relatively long arms with a relatively short scapula, a rounded thorax with a small diameter, a highly mobile sterno-clavicular articulation, and emphasis on powerful flexion in the proximal limb joints via advantageous lever arms. Despite these changes, patterns of limb kinematics remained conservative during the course of evolution in the lineages leading to extant tree sloths, and it is suggested here that this also applies to the pattern of neuromuscular control of limb movements during locomotion. Morphological ‘solutions’ to altered functional demands posed by inversed orientation of the body differ in the two genera of extant tree sloths, thereby corroborating the proposed diphyly. Convergent evolution in tree sloths may be attributed to functional constraints posed by fossorial adaptations in early Xenarthra that canalized sloths to adopt a suspensory posture and locomotion in the arboreal habitat.     

Immobilization of free-ranging Hoffmann's two-toed and brown-throated three-toed sloths using ketamine and medetomidine: a comparison of physiologic parameters

Free-ranging Hoffmann's two-toed sloths (Choloepus hoffmanni; n=26) and brown-throated three-toed sloths (Bradypus variegatus; n=15) were manually captured and immobilized with 2.5 mg/kg ketamine + 0.02 mg/kg medetomidine administered intramuscularly. Physical examinations were conducted on each sloth 10 min after initial injection, and blood, fecal, and ectoparasite samples were collected. Heart rate, respiratory rate, body temperature, indirect systolic blood pressure, and indirect peripheral oxygen saturation were monitored every 5 min for the duration of anesthesia. After 45 min, atipamazole (0.1 mg/kg) was administered intramuscularly, as an antagonist to medetomidine, in order to facilitate recovery. All recoveries were smooth, rapid, and uneventful. Physiologic parameters were compared across time, gender, and species. All sloths, regardless of species and gender, demonstrated a time-dependent decrease in heart rate and blood pressure, and an increase in respiratory rate, during the course of anesthesia. Peripheral oxygen saturation was similar for all sloths over time. There were significant species differences for heart rate (Choloepus > Bradypus), respiratory rate (Choloepus > Bradypus), and systolic blood pressure (Bradypus > Choloepus), while there were significant gender differences for body temperature (males > females) and blood pressure (males > females). Results of this study suggest that the ketamine-medetomidine mixture, as described above, is a safe and effective anesthetic combination in free-ranging two- and three-toed sloths, although peripheral blood pressure should be monitored during anesthesia.     

Spatial ecology and conservation of two sloth species in a cacao landscape in limón,Costa Rica

The spatial ecology of sloths was studied in an agricultural landscape in Limón Province, Costa Rica. Two sloth species, the three-toed sloth (Bradypus variegatus) and the two-toed sloth (Choloepus hoffmanni), actively used and traveled through a cacao agroforest and its contiguous living fence rows and riparian forests. This agroecosystem was embedded in an agricultural landscape dominated by banana and pineapple plantations and pastures with dispersed trees. The two-toed sloth (C. hoffmanni) was found in 101 tree species and used 34 for food; the three-toed sloth (B. variegatus) was found in 71 tree species and used 15 for food. Choice of preferred species differed between the two sloth species. Trees commonly used by sloths for food and/or refuge in the cacao agroforest included Erythrina poeppigiana, Cecropia obtusifolia, Leucaena leucocephala; in the living fence rows, Cordia alliodora, Erythrina poeppigiana, Ocotea sinuata and Trophis racemosa; in the riparian forests, Coussapoa villosa, Cecropia obtusifolia, Hura crepitans, Pterocarpus officinalis and Spondias mombin; and in the pastures with dispersed trees, Cordia alliodora, Coussapoa villosa, Erythrina poeppigiana, Ocotea sinuata and Hura crepitans. This study demonstrates the importance of the cacao agroforest as well as arboreal elements in other land uses in providing resources for sloth conservation in a larger agricultural landscape.     

Limited performance of DNA barcoding in a diverse community of tropical butterflies

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.     

Estimation of hearing capabilities of Early Miocene sloths (Mammalia,Xenarthra, Folivora) and palaeobiological implications

The Miocene Santacrucian sloths are a very important assemblage because they represent the first major radiation among sloths and, therefore, they could provide many insights about sloth evolution and diversity. Based on other studies, the hearing capabilities of Pleistocene sloths were identified. For a deeper understanding of these capabilities from an evolutionary point of view, in this article we study the hearing capabilities of Santacrucian sloths. We estimate the frequency range and the best frequency of hearing of six species of stem-megatherines and one species of mylodont. In most cases, the best frequency of hearing is much lower than the expected value for the estimated body mass of the species. This general shift towards low values of frequency is different than the pattern observed in mammals where sound localisation is improved by an increased sensitivity to high frequencies. In this article, we also propose some palaeobiological inferences of the low-frequency shift of hearing range in Miocene Santacrucian sloths.     

Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato

Reliable and easy to use techniques for chromosome identification are critical for many aspects of cytogenetic research. Unfortunately, such techniques are not available in many plant species, especially those with a large number of small chromosomes. Here we demonstrate that fluorescence in situ hybridization (FISH) signals derived from bacterial artificial chromosomes (BACs) can be used as chromosome-specific cytogenetic DNA markers for chromosome identification in potato. We screened a potato BAC library using genetically mapped restriction fragment length polymorphism markers as probes. The identified BAC clones were then labeled as probes for FISH analysis. A set of 12 chromosome-specific BAC clones were isolated and the FISH signals derived from these BAC clones serve as convenient and reliable cytological markers for potato chromosome identification. We mapped the 5S rRNA genes, the 45S rRNA genes, and a potato late blight resistance gene to three specific potato chromosomes using the chromosome-specific BAC clones. Received: 19 January 2000 / Accepted: 27 March 2000     

The wrist joint of two-toed sloths and its relevance to brachiating adaptations in the hominoidea

Two-toed sloths have evolved a wrist complex that includes the following traits: (1) diminution and distal migration of the pisiform, with a loss of contact with the ulna; (2) reduction of the distal end of the ulna to a styloid process; and (3) extremely reduced contact between the ulna and triquetrum. These traits were proposed by Lewis ('65, '74) to be indicative of brachiating habits and to be a unique adaptation of the Hominoidea. Cartmill and Milton ('77) recently found a similar complex in the wrists of the lorisines. Very similar adaptations of the wrist among the Hominoidea, lorisines, and two-toed sloths clearly refute contentions of Lewis and strengthen the hypothesis of Cartmill and Milton that the traits common to those animals are due to similar slow, cautious, but acrobatic locomotion.     

Isolation of a sex-linked DNA sequence in cranes.

A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.     

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.     

Sleeping outside the box: electroencephalographic measures of sleep in sloths inhabiting a rainforest

The functions of sleep remain an unresolved question in biology. One approach to revealing sleep's purpose is to identify traits that explain why some species sleep more than others. Recent comparative studies of sleep have identified relationships between various physiological, neuroanatomical and ecological traits, and the time mammals spend in rapid eye movement (REM) and non-REM sleep. However, owing to technological constraints, these studies were based exclusively on animals in captivity. Consequently, it is unclear to what extent the unnatural laboratory environment affected time spent sleeping, and thereby the identification and interpretation of informative clues to the functions of sleep. We performed the first electroencephalogram (EEG) recordings of sleep on unrestricted animals in the wild using a recently developed miniaturized EEG recorder, and found that brown-throated three-toed sloths (Bradypus variegatus) inhabiting the canopy of a tropical rainforest only sleep 9.63 h d(-1), over 6 h less than previously reported in captivity. Although the influence of factors such as the age of the animals studied cannot be ruled out, our results suggest that sleep in the wild may be markedly different from that in captivity. Additional studies of various species are thus needed to determine whether the relationships between sleep duration and various traits identified in captivity are fundamentally different in the wild. Our initial study of sloths demonstrates the feasibility of this endeavour, and thereby opens the door to comparative studies of sleep occurring within the ecological context within which it evolved.     

Identification and cloning of Lmairk, a member of the Aurora/Ipl1p protein kinase family, from the human protozoan parasite Leishmania.

Lmairk, a gene encoding a member of the Aurora/Ipl1p family of protein kinases (AIRK), was cloned from the protozoan parasite Leishmania major. Aurora kinases are key enzymes involved in the regulation of normal chromosome segregation during mitosis and cytokenesis of eukaryotic cells. This single-copy gene located on L. major chromosome 28 encodes a 301 amino acid polypeptide. All 11 conserved eukaryotic protein kinase catalytic subdomains are present and the proposed AIRK signature sequence was identified in the activation loop between subdomains VII and VIII. Lmairk is expressed, as an approximately 2.4 kb message, in at least three different species of Leishmania. This report represents the first identification of an AIRK from the trypanosomatid family of early divergent eukaryotes.     

Crested gibbon (Hylobates [Nomascus]) identification using noninvasively obtained DNA

The development of captive breeding programs for the crested gibbons (Hylobates [Nomascus]) of Indochina is hindered by the difficulty of sorting individuals into their correct species and subspecies groups. We describe taxon-specific DNA sequence variation in a 252-base pair region of the mitochondrial cytochrome b gene, which can be used to identify individuals of the three taxa of crested gibbon commonly found in Western zoos. These molecular genetic markers can be amplified from plucked hair and will permit the identification of morphologically similar females and crested gibbons of either sex of unknown origin. Noninvasive genotyping of captive animals will facilitate the genetic management of these critically endangered primates. © 1994 Wiley-Liss, Inc.     

Restriction fragment length polymorphisms of a chloroplast photosystem II gene from poplar and their use for species identification.

In a total DNA library from the poplar clone Beaupré (Populus trichocarpa x P. deltoides) one DNA clone was found to identify restriction site polymorphisms in different poplar species. This clone represents a cpDNA gene that shows close homology to a photosystem II gene of pea and spinach coding for the D2 protein and the 44 kDa reaction centre. In Southern blot analysis this probe identified interspecific restriction site variation among the different poplar species; intraspecific variation was not detectable. As the chloroplast genome is maternally inherited in poplars this cpDNA probe was used for identification of P. nigra or P. deltoides as the seed parents of F1 hybrid trees in natural stands of western Germany.