Highly repeated DNA analysis and systematics of the Lemuridae,a family of Malagasy prosimians

Hybridization of highly repeated DNA sequences ofEulemur fulvus mayottensis, Lemur catta, andVarecia has been performed on blots of different species of Lemuridae (L. catta, Hapalemur griseus, Varecia variegata variegata, V. v. rubra, E. macaco macaco, E. coronatus, E. mongoz, andE. rubriventer). The probe ofE. fulvus only hybridized with the differentEulemur species, whereas that ofVarecia hybridized with the two subspecies ofVarecia and that ofL. catta with bothL. catta andHapalemur. These results were used to confirm the classification ofVarecia in a separate genus and to review the separation of theL. catta/Hapalemur group from the other species ofEulemur. Comparison of the migration patterns from DNA fragments of these different species has been used to propose a cladogram of the differentEulemur species.     

Development, structure and function of rhinoceros enamel

Vertical enamel prism decussation in the inner-layer enamel of rhinoceroses occurs as the result of vertical translation, in opposite senses, of zones of ameloblasts, which begins very shortly after amelogenesis commences at the enamel-dentine junction. Prisms in the centre of the decussating zones are stacked in the Pattern 3 arrangement. Zone boundary prisms adopt intermediate orientations, are locally nearly perpendicular to the enamel surface, and have a cylindrical, Pattern 1 cross-section. Decussation also continues in the outer-layer enamel, but the prisms all have occlusal-going courses: the occlusal-going zones of the inner enamel continue as the more occlusally oriented zones of the outer layer. Abrasion resistance to diamond polishing and soft abrasive projectile erosion (air-polishing with NaHCOs) and resistance to ion beam erosion is greater with distance from the nearest prism boundary discontinuity. Polished surface areas containing longitudinally sectioned prisms are more prone to 'air-polishing' and 'airbrading' erosion than areas with transversely sectioned prisms. These observed relationships fully explain the relief developed at natural wear surfaces.     

Highly repeated DNA sequences and systematics of malagasy primates

We have analyzed highly repeated DNA sequences of Malagasy lemurs with restriction enzymes, Southern blotting and nucleotide sequencing to assess relatedness among repeated DNA fragments from different species. We have focused our studies on two prosimian families:Lemuridae andCheirogaleidae. Our results have allowed us to draw the following conclusions: confirmation of the separation based on molecular biology data ofEulemur species from theLemur catta/Hapalemur group; classification ofVarecia in a genus clearly separated from the otherLemuridae genera; confirmation of the specific status ofHapalemur aureus; confirmation of cytogenetic data in favour of the subdivision of the familyCheirogaleidae into two subfamilies:Cheirogaleinae andPhanerinae. Finally a cladogram has been constructed by numbering all the highly repeated DNA fragments (obtained by Southern blotting) and analyzing the inferred systematic relationships between the Lemuridae and Cheirogaleidae species.     

The prism pattern of rat molar enamel: a scanning electron microscope study.

Rat molar enamel has been studied by sectioning the enamel along various planes, and observing the etched surfaces in the SEM. It was found that the prism pattern was much more variable than in rat incisor enamel. Regions without prism decussation seemed to dominate in the occlusal half of the molars. Where present, prism decussation was of the uniserial lamellar type, but it varied considerably in distribution, extent, and distinctness. Prism decussation seemed to have a predilection for the cervical enamel, and was almost absent in the enamel on the occlusal surface. The interprismatic substance showed a characteristic configuration: In the inner enamel it appeared in the form of radially oriented sheets, which tended to delimit radially directed, single lines of prisms. In regions with prism decussation these single lines of prisms encompassed prisms belonging to different prism lamellae. In the outer part of the enamel the interprismatic substance exhibited a honeycomb appearance. The similarities and differences between the prism patterns of rat incisor and molar enamel may be of importance for understanding the mechanisms of amelogenesis, especially for the recognition of factors controlling the movement of ameloblasts.     

Chromosome variability in the Lemuridae

Chromosome studies have been conducted in Lemur catta, L. macaco, L. mongoz, L. fulvus fulvus, and a hybrid L. fulvus fulvus x L. fulvus albifrons. Comparative analysis shows that inter- and intraspecific chromosome variability is a common finding in Lemurs. The Lemuridae are divided into three groups according to the characteristics of their chromosome complement. A reclassification of L. catta with the Hapalemurs is suggested, based on chromosomal and nonchromosomal data.     

Microstructural Reinforcement in the Canine Enamel of the Hyaenid Crocuta crocuta, the FelidPuma concolorand the Late Miocene Canid Borophagus secundus

In bone-eating carnivores such as the hyena Crocuta crocuta, the tooth enamel contains a secondary vertical prism decussation phyletically derived from the wavelike horizontal decussation of primitive carnivores. The structure resists fracture under vertical, oblique, and horizontal tensile stresses, owing to the following modifications of the primitive structure. Positions of wave crests and of wave troughs are synchronized in the vertically successive layers of decussating prisms. Prisms in each successive layer run in a common direction at the crests and in a common but reversed direction at the troughs. Between the crests and troughs, prisms in obliquely slanting layers often retain their primitively reversed prism directions. Near the enamel–dentine junction (EDJ), irregular horizontal decussation is retained. In the upper canine of C. crocuta, a consumer of large bones, secondary vertical decussation is largely confined to the labial and anterior sides of the crown toward the tip where modeling of the static stresses predicts the tensile stresses to be highest and aligned vertically. In Puma concolor, which does not consume large bones, secondary vertical decussation is absent, indicating stress magnitude to be a critical factor in the selection for secondary vertical decussation. The canine enamel in Borophagus secundus, an extinct canid with derived aspects of skull and dental shape like those in hyenas, has dental structures similar to those in C. crocuta but which differ in several ways. The wavelike shapes of the decussation planes are better developed in transverse sections in B. secundus than in C. crocuta, suggesting either the folds are less modified or they dip at a steeper angle. Secondary vertical decussation in B. secundus is more extensive around the circumference of the canine than in C. crocuta, related to a difference in cross-sectional shape of the tooth. Vertical prism decussation may have been more frequently attained in carnivorous mammals than in ungulates because of the more random orientation of dental stresses which creates a selective advantage for wavy decussation planes—a structural transition to vertical decussation.     

Enamel structure and microwear: an experimental study of the response of enamel to shearing force.

The anisotropic fracturing and differential wear properties of enamel microstructure represent factors that can obscure the predictive relationship between dental microwear and diet. To assess the impact of enamel structure on microwear, this in vitro experimental study examines the relative contributions to wear of three factors: 1) species differences in microstructure, 2) direction of shearing force relative to enamel prisms and crystallites, and 3) size of abrasive particles. Teeth of Lemur, Ovis, Homo, and Crocodylus, representing, respectively, the structural categories of prismatic patterns 1, 2, and 3 and nonprismatic enamel, were abraded by shearing forces (forces having a component directed parallel to abraded surfaces) and examined by scanning electron microscopy. Striation width increased with particle size for nonprismatic, but not for prismatic, specimens. Direction of shear relative to prism and crystallite orientation had a significant influence on striation width in only some prismatic enamels. The different responses of prismatic and nonprismatic enamels to abrasion reflect the influence of structure, but at the level of organization of crystallites rather than prisms per se. Such interactions explain in part the inability of striation width to discriminate among animals with different dietary habits. Heteroscedasticity and deviations from normality also may confound parametric analyses of microwear variables. Variation in crystallite orientation in prismatic enamels may contribute to optimal dental function through the property of differential wear in functionally distinct regions of teeth.     

The banded chromosomes of coquerel’s sifaka,Propithecus verreauxi coquereli (Primates,Indriidae)

The karyotype of Propithecus verreauxi coquereli isdescribed in this report using G-, Q-, and C-banding and silver staining for nucleolus organizer regions. The banded chromosomes of P. v. coquereli(family Indriidae) are compared with those of Lemur fulvus fulvus(family Lemuridae), revealing few karyotypic similarities between the two groups and suggesting a wellestablished phylogenetic divergence between these families.     

Phylogenetic relationships among Malagasy lemurs as revealed by mitochondrial DNA sequence analysis

Systematics and evolution of Malagasy lemurs has been analyzed using morphological characters, fossil evidence, ecological/ethological data, and chromosomal banding patterns. Recent developments in DNA technology have provided evolutionary biologists with additional and powerful tools for making phylogenetic inference. In the last years several studies concerning highly repeated DNA sequences (hrDNA) provided new insights about the systematic relationships among the different species of Lemuridae and Cheirogaleidae. Here, a reconstruction of molecular phylogeny of extant Malagasy lemurs based on the comparison of cytochrome-b mitochondrial DNA sequences is presented. With the Polymerase Chain Reaction (PCR) and direct sequencing of amplified DNA fragments, both the phylogenetic range and resolving power of comparative analysis can be extended. These techniques allow to gather sequence data useful to evaluate the pattern of molecular evolution offering opportunities for phylogenetic purposes. A 290-bp fragment of cytochrome-b gene has been amplified and sequenced from the following species:Tupaia glis, Galago alleni, Daubentonia madagascariensis, Indri indri, Varecia variegata, Eulemur fulvus, Eulemur coronatus, Eulemur rubriventer, Eulemur mongoz, Eulemur macaco, Lemur catta, andHapalemur griseus griseus. The phylogenetic trees obtained show the relationships among the Eulemur species and confirm the karyological and hrDNA results of a separated clade forL. catta/Hapalemur. The separation ofVarecia variegata from the other genus of the family Lemuridae is discussed.     

Enamel microstructure and molar wear in the greater galago,Otolemur crassicaudatus (mammalia,primates)

This study describes the molar enamel microstructure of the greater galago, based on SEM study of four individuals. Galago molar enamel consists primarily of radially oriented Pattern 1 prisms. However, the most superficial enamel is characterized by regions of poorly developed prisms or nonprismatic enamel, and Pattern 3 prisms can be found at depths intermediate and deep to the enamel surface. Orientations of prism long axes relative to wear surfaces differ among functionally distinct regions (cuspal facets, Phase I/II facets, and crushing basins). Consequently, orientations of enamel crystallites relative to these surfaces also differ. Because crystallites are the structural unit involved in enamel abrasion, these differences in orientation may have important effects on molar wear patterns. Crystallite orientations differ most between cuspal facets and Phase I/II facet surfaces. Cuspal facets are characterized by near surface-parallel interprismatic and surface-oblique prismatic crystallites. Previous experimental studies suggest that this arrangement is most resistant to wear when surface-normal (compressive) loads predominate. In contrast, prismatic and interprismatic crystallites intercept Phase I/II facet surfaces obliquely, an arrangement expected to resist abrasion when surface-parallel (shearing) loads predominate. Superficial enamel is preserved at most basin surfaces, indicating that these regions are subject to comparatively little abrasive wear. These results support the hypothesis that galago occlusal enamel is organized so as to resist abrasion of different functional regions, a property that may prove important in maintaining functional efficiency. However, this largely reflects constraints of occlusal topography on a microstructure typical of many mammals and thus does not appear to represent a structural innovation. © 1993 Wiley-Liss, Inc.     

Molecular biology and systematics of an extinct Lemur:Pachylemur insignis

The systematic position of the extinctPachylemur insignis has been controversial: some authors consideredPachylemur as a lemur, whereas others viewed it closer toVarecia. Its classification in the genusLemur orVarecia thus remained an open question. DNA extraction from subfossil bones, using a non-destructive method, allowed us to obtain enough material to make a Southern blot. The hybridization ofPachylemur withEulemur fulvus, Lemur catta, andVarecia variegata highly repeated DNA probes showed that only theVarecia probe gave a positive signal on hybridization on thePachylemur blot. These results indicate thatPachylemur must be considered closer to the genusVarecia than toEulemur andLemur.     

Milk composition reflects pattern of material care in prosimian primates

Hypothesized relationships between milk composition and life history traits were examined by analyzing mid-lactation milks of seven lemurs (Eulemur fulvus, E. macaco, E. rubriventer, E. mongoz, Varecia variegata, Hapalemur griseus, Lemur catta), three bushbabies (Otolemur crassicaudatus, O. garnettii, Galago moholi), and two lorises (Nycticebus coucang, Loris tardigradus); partial data were also obtained for the lemuroid Cheirogaleus medius. There were significant differences in milk composition among species within either Eulemur or Otolemur, but the four genera for which multiple samples were available (Eulemur, Varecia, Otolemur, and Nycticebus) exhibited large composition differences. Eulemur milk was, on average, very dilute (9.9% dry matter) and low in energy (0.49 kcal/g). These milks contained 0.9% fat, 1.2% protein, and 8.4% carbohydrate on a fresh weight basis. Protein energy comprised only about 15% of total milk energy. Varecia had significantly higher dry matter (13.5%), fat (3.2%), protein (4.2%), gross energy (0.80 kcal/g), and protein energy: total energy ratio (28%) than Eulemur. Milks of the lorisoid genera Otolemur and Nycticebus were very similar, and both had significantly higher dry matter (18.3, 16.3%), fat (7.6, 7.0%), and gross energy concentration (1.27, 1.13 kcal/g) than either lemuroid genus. Otolemur milk was higher in protein than Nycticebus milk. We conclude that lorises, bushbabies, and perhaps cheirogaleids produce relatively rich, energy-dense milks in comparison with anthropoid primates. However, dilute milks appear to be uniformly found among species of Eulemur and perhaps in Lemur catta. The milk of Varecia (and perhaps Hapalemur) is intermediate in composition. Differences in milk composition among prosimians may be related to differences in maternal care: prosimians that carry their young during lactation produce more dilute milks than do species which leave their young unattended for prolonged periods. When looking at primates as a whole, however, the picture is somewhat less clear, since the milks of some “parkers” like Varecia do closely resemble those of large anthropoid primates who carry their young. Am. J. Primatol. 41:195–211, 1997. © 1997 Wiley-Liss, Inc.     

Phylogeny and character behavior in the family Lemuridae

A phylogenetic analysis of the family Lemuridae was accomplished using multiple gene partitions and morphological characters. The results of the study suggest that several nodes in the lemurid phylogeny can be robustly resolved; however, the relationships of the species within the genus Eulemur are problematically nonrobust. The genus Varecia is strongly supported as the basal genus in the family. Hapalemur and Lemur catta are strongly supported as sister taxa and together are the sister group to the genus Eulemur. E. mongoz is the most basal species in the genus Eulemur. E. fulvus subspecies form a monophyletic group with three distinct lineages. E. coronatus is strongly supported as the sister taxon to E. macaco. The relationships of E. rubriventer, E. fulvus, and the E. macaco-E. coronatus pair are unresolved. Our combined molecular and morphological analysis demonstrates the lack of influence that morphology has on the simultaneous analysis tree when these two kinds of data are given equal weight. The effects of several extreme weighting schemes (removal of transitions and of third positions in protein-coding regions) and maximum-likelihood analysis were also explored. We suggest that these other forms of inference add little to resolving the problematic relationships of the species in the genus Eulemur.     

A possible role of plantations for primate conservation in Madagascar

The utilization of eucalyptus plantations by seven sympatric species of prosimians was studied in the eastern rainforest of Madagascar. The species were Avahi laniger, Cheirogaleus major, Hapalemur griseus, Indri indri, Lemur fulvus, Lepilemur mustelinus, and Microcebus rufus. None of the lemurs was ever found in young eucalyptus plantations with little undergrowth. This was mainly due to the lack of travel opportunities within the shrub layer and between the shrubs and the canopy. Food (mainly berries) is seasonally available in the shrub layer but cannot be exploited because frugivorous lemurs cannot reach it. Old eucalyptus plantations with dense undergrowth are used by all prosimian species. They provide food as well as travel and resting facilities. Mixed tree plantations in the western part of Madagascar were used by groups of Lemur fulvus, Lepilemur mustelinus, and Propithecus verreauxi. According to these results, old eucalyptus plantations and mixed tree species plantations could be used to provide firewood and construction wood for the human population. They also might extend the habitat for lemurs and serve as buffers against human disturbance.     

Strepsirhine dichotomy from a human perspective (Primates: Lorisoidea, Lemuroidea)

Using monospecific, polyclonal antisera against 69 human plasma proteins, 128 antigenic determinants from 40 cross-reacting homologues were characterized in representatives of the prosimian genera Lemur, Eulemur, Varecia (Lemuridae) and Otolemur (Galagidae). Seventeen determinants from 16 different proteins were absent in homologues of the gagalo but were shared by lemurs and several platyrrhines, cercopithecids, and hominoids. Smaller locus samples for potto(Perodicticus potto) and slow loris (Nycticebus coucang) confirmed the more distant immunological relationship of lorises than of lemurs to anthropoids. If evolutionary rates are constant and equal in lorisiform and lemuriform prosimians, this patern of character distribution indicates strepsirhine paraphyly, lorises diverging earlier (possibly some 6 times 10⁶ years) than lemurs from anthropoid ancestors. If this is so, lorises rather than true lemurs should be elected to root the polarity of character evolution in Primates. As an alternative, galagine proteins evolve more rapidly than lemurine homologues.     

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus,Wallabia bicolor and Peradorcas concinna)

Summary Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 m in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.     

Phylogenetic relationships among Lemuridae (Primates): evidence from mtDNA

The family Lemuridae includes four genera: Eulemur, Hapalemur, Lemur,Varecia. Taxonomy and phylogenetic relationships between L. catta, Eulemur and Hapalemur, and of Varecia to these other lemurids, continue to be hotly debated. Nodal relationships among the five Eulemur species also remain contentious. A mitochondrial DNA sequence dataset from the ND 3, ND 4 L, ND 4 genes and five tRNAs (Gly, Arg, His, Ser, Leu) was generated to try to clarify phylogenetic relationships w ithin the Lemuridae. Samples (n=39) from all ten lemurid species were collected and analysed. Three Daubentonia madagascariensis were included as outgroup taxa. The approximately 2400 bp sequences were analysed using maximum parsimony, neighbor-joining and maximum likelihood methods. The results support monophyly of Eulemur, a basal divergence of Varecia, and a sister-group relationship for Lemur/Hapalemur. Based on tree topology, bootstrap values, and pairwise distance comparisons, we conclude thatVarecia and Eulemur both represent distinct genera separate from L. catta. H. griseus andH. aureus form a clade with strong support, but the sequence data do not permit robust resolution of the trichotomy involving H. simus, H. aureus/H. griseus and L. catta. Within Eulemur there is strong support for a clade containing E. fulvus, E. mongoz and E. rubriventer. However, analyses failed to clearly resolve relationships among those three species or with the more distantly related E. coronatus and E. macaco. Our sequencing data support the current subspecific status of E.m. macaco and E.m. flavifrons, and that of V.v. variegata and V.v. rubra. However, tree topology and relatively large genetic distances among individual V.v. variegata indicate that there may be more phylogenetic structure within this taxon than is indicated by current taxonomy.     

Shape of the lateral mandibular outline in Lemuridae: a quantitative analysis of variability using elliptical Fourier analysis

While several morphometric analyses in lemurids have focused on the craniofacial complex, the characterization of their mandibular morphology has received less attention. The mandibular outline, in lateral perspective, was quantified using elliptical Fourier analysis, in an osteological sample encompassing 189 lemurid mandibles (66 Eulemur, 51 Hapalemur, 22 Lemur and 50 Varecia), and compared using multivariate statistical techniques. The taxonomic value of this outline in Lemuridae was demonstrated by the existence of significant separations between the four genera studied. In particular, the mandibular morphology of Hapalemur was markedly different from that in the group Eulemur-Lemur-Varecia. Excluding Hapalemur from analysis, the distinctions between Eulemur, Lemur and Varecia were enhanced suggesting the existence of more subtle intergeneric differences in mandibular morphology. Variation in mandibular form was greatest in Hapalemur and smallest in Eulemur and Varecia (as demonstrated by the mean values of interindividual distances); variation was higher in Lemur than in Eulemur and Varecia, but not higher than in Hapalemur. This morphological diversity may be related to functional adaptation in response to particular dietary habits. The patterns of intergeneric and intrageneric shape variations of the mandible in Lemuridae presented here provide a valuable resource for the analysis of variation among living and fossil lemurids.     

Phylogenetic Affinities Among the Extant Malagasy Lemurs (Lemuriformes) Based on Morphology and Behavior

The difficulty in achieving a consensus on the phylogenetic relationships of lemuriform primates has been due largely to the lack of a lemur fossil record and to the lack of an appropriate outgroup that would facilitate polarization of character states. Recent findings allow us to polarize some of the bony characters, but to a large extent this problem still remains. In the past, phylogenetic analyses have focused on specialized character sets such as dentition or basicranial traits, or they have employed differential weighting schemes to a more variable set of characters. In the analysis presented here, I combined all relevant characters available in the literature into one data set but restricted my selection to those traits having discontinuous states and for which no contradictory coding schemes were published. I reduced the assumptions in this analysis by removing most external weighting and ordering effects on these data sets. The available data from the literature were supplemented with data from my own observations at the Duke University Primate Center. Data were collected for 25 characters and 20 taxa and were submitted to a cladistic analysis. Some important findings from this study include support for (1) a sister-group relationship between Lepilemur and the Indridae, (2) a sister-group relationship between the Lemuridae (except Varecia) and the Indridae/Lepilemur clade, (3) a monophyletic genus Eulemur, and (4) the exclusion of Varecia from the Lemuridae.