Localization of a new highly repeated DNA sequence of Lemur cafta (Lemuridae, Strepsirhini).

We have isolated and cloned an 800-bp highly repeated DNA (HRDNA) sequence from Lemur catta (LCA) and described its localization on LCA chromosomes. Lemur catta HRDNA sequences were localized by performing FISH experiments on standard and elongated metaphasic chromosomes using an LCA HRDNA probe (LCASAT). A complex hybridization pattern was detected. A strong pericentromeric hybridization signal was observed on most LCA chromosomes. Chromosomes 7 and 13 were lit in pericentromeric regions, as well as in the interspersed heterochromatin. Chromosomes 1, 3, 4, 17, 19, X, and microchromosomes (20, 25, 26, and 27) showed no signals in the pericentromeric region, but chromosomes 3 and 4 showed a positive hybridization in heterochromatic regions. The 800-bp L catta HRDNA was species specific. We performed FISH experiments with the LCASAT probe on Eulemur macaco macaco (EMA) and Eulemur fulvus fulvus (EFU) metaphases and no positive signal of hybridization was detected. These findings were also confirmed by Southern blot analysis and PCR.     

Confirmation, via in situ hybridization, of the occurrence of Robertsonian translocations during lemur evolution by localization of GLUDP1 DNA sequences on lemur chromosomes.

A human genomic DNA sequence derived from glutamate dehydrogenase pseudogene 1 was used as a probe for in situ hybridization to the chromosomes of three lemur species, Eulemur fulvus mayottensis (EFU), E. macaco macaco (EMA), and E. coronatus (ECO). This sequence, which is 98% homologous to the nucleotide sequence of the gene for human glutamate dehydrogenase (GLUD), was found on homologous bands of three morphologically similar chromosome segments, EFU14, EMA5p, and ECO8q, confirming that different Robertsonian translocations occurred during the evolution of these three species. These loci on the lemur chromosomes probably correspond to the human GLUD locus.     

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.     

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.     

Relationships among brown lemurs (Eulemur fulvus) based on mitochondrial DNA sequences

The brown lemurs (Eulemur fulvus) include seven subspecies, whose evolutionary relationships remain contentious. In particular, it is unclear whether the Malagasy and Comorian E. f. fulvus populations are differentiated at the subspecific level (E. f. mayottensis). Furthermore, it has been suggested that E. f. collaris and E. f. albocollaris are separate species. Analyses of approximately 2400 bp of mitochondrial DNA sequence data from part of the COIII gene, together with complete genes for ND3, ND4L, and ND4 and 5 tRNAs, resolved 34 E. fulvus specimens into six clades: ((albocollaris, collaris) (rufus (rufus (fulvus/mayottensis (albifrons/fulvus/sanfordi))))). Based on the information available and our analyses we conclude that E. f. albocollaris and E. f. collaris do not represent species distinct from E. fulvus and that Comorian brown lemurs do not deserve subspecific rank. No genetic differentiation was detected between E. f. albifrons and E. f. sanfordi; on the other hand, there are obviously two separate lineages of E. f. rufus.     

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.     

Habitat use and social structure of a brown lemur hybrid population in the Berenty Reserve, Madagascar

The population of brown lemurs has rapidly grown since their founders were introduced to the Berenty Reserve. The founders consist of two species (Eulemur fulvus rufus and E. collaris). To characterize the behavior of the population and to examine whether these characteristics affect population growth, I investigated the habitat use and social structure of the population of brown lemurs at Berenty (Berenty Eulemur). Behavior data were collected focusing on horizontal and vertical habitat use, activity rhythms, and intergroup relationships. These data were compared with the data of E. fulvus in other areas, with the previous studies done at Berenty, and with data on Berenty Lemur catta. Berenty Eulemur maintained a home range size comparable to E. f. rufus in the western deciduous dry forest, but was found at a lower level of the forest and had larger overlapping home ranges. Berenty Eulemur use food resources earlier in the morning than L. catta, intergroup conflict was avoided by vocal communication, and Berenty Eulemur made suitable use of their limited habitat. I suggest that a number of behavioral characteristics of Berenty Eulemur may contribute to their population growth.     

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.     

Species concepts and the determination of historic gene flow patterns in the Eulemur fulvus (Brown Lemur) complex

Eulmur fulvus , a complex comprising six subspecies, is a classic example of species status conferred through evolutionary taxonomy. We used the phylogcnctic species concept as an alternative method to the biological species concept for determining historic patterns of gene flow between the various E. julvus subspecies and for conferring species status. In this paper, we used population aggregation analysis to determine the proper species partitions and cladistic analysis to reconstruct the evolutionary relationships of the different populations in the Eulemur fulvus complex. We sequenced three mtDNA gene regions (d-loop, 12S, and cyt-b) and one nuclear region, casein kinase, for a total of 1247 bases. Through population aggregation analysis, we determined that the E. fulvus complex should be split into three units; one unit supported by six diagnostic sites comprising E.f. albocollans , one unit supported by three diagnostic sites comprising E.f. collaris , and one unit supported by two diagnostic sites comprising the four other subspecies. Although all six subspecies in the E. julvus complex share a common ancestor, we found in our cladistic analysis that E. J. collaris and E. j. albocollans share a common ancestor that more recently split off from the common ancestor of the four other E. fulvus subspecies.     

Genetic assessment of a white-collared x red-fronted lemur hybrid zone at Andringitra,Madagascar

We examined a purported lemur (Eulemur fulvus rufusxE. albocollaris) hybrid zone at Andringitra, Madagascar, using sequences from five genes (one mitochondrial gene (d-loop) and four nuclear introns (hemopexin, malic enzyme, ceruloplasmin, and microsatellite 26 flanking region)), from 60 individuals (E. albocollaris (n = 16), E.f. rufus (n = 14), E. collaris (n = 9), and purported hybrids from Andringitra (n = 21)). Diagnostic (d-loop and microsatellite 26) and private sites (all other genes) were found in all gene regions for E. albocollaris and E.f. rufus. Also, private sites were found for the purported hybrid population in two gene regions (d-loop and ceruloplasmin). When the putative hybrids were examined for diagnostic and private markers, 18 of 21 were found to contain markers from both E. albocollaris and E.f. rufus populations. The remaining three individuals were found to contain only markers for E. albocollaris. These results indicate that the population at Andringitra is a hybrid population between E. albocollaris and E.f. rufus.     

Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.

In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.     

Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome

The LMR1 5.2 kb interspersed repeat of Leptosphaeria maculans was described by Taylor and Borgmann [Mol. Plant Microbe Interact. 7 (1994) 181] as an uncharacterized repeated element sharing homologies with both LINEs and SINEs. Here, we used the LMR1 sequence as a template to identify the full-length element within a 184-kb genomic sequence corresponding to the pericentromeric region of the 2.80 Mb chromosome of isolate v23.1.3. This region comprises (i) one 6980-bp full-sized Pholy element bordered by two 275- to 280-bp long terminal repeats (LTRs), (ii) five Pholy-related sequences, usually truncated at their 3' ends, and (iii) five solo-LTRs. Structural features strongly suggested that Pholy corresponds to an ancient copia-like retrotransposon, sharing strong homologies with the Elsa retrotransposon of Stagonospora nodorum. Pholy was also suggested to be specific to pericentromeric regions. Comparative analysis of the structure of the Pholy-like sequences occurring in the 184-kb contig and in other parts of the genome showed that this family of repeats is highly degenerated following extensive repeat induced point mutation (RIP).     

The concept of cathemerality: history and definition

During a field study in 1974 it was noticed, and further fieldwork in 1977 and 1980 confirmed, that the activity of the Mayotte lemur, Eulemur fulvus fulvus, is distributed fairly evenly throughout the daily 24-hour cycle: by a very crude approximation the daytime activity:rest ratio averages 0.271, while the night-time ratio averages 0.283. In 1978 I proposed the new word 'cathemeral', compounded from the Greek roots kappaalphataualpha (through) and etamuepsilonrhoalpha (the day), to describe this unusual activity pattern, though for reasons described in this article the term was not formally published until 1988. Since then cathemeral activity has been routinely recorded, with some variation, in species of the strepsirhine genera Eulemur and Hapalemur; and in one form or another it apparently also occurs, at least occasionally, among platyrrhines (species of Aotus and Alouatta) as well as among a variety of non-primate mammals. It may well thus be a more widespread activity pattern than earlier appreciated.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin.     

Census of brown lemurs (Eulemur fulvus sspp.) in southeastern Madagascar: Methods‐testing and conservation implications

Four forest areas were censused in southeastern Madagascar from June–August 1995 to estimate local population densities and habitat conditions for two threatened subspecies of brown lemur (Eulemur fulvus collaris and Eulemur fulvus albocollaris). Survey transects varied in length (1–3.5 km) and in surveillance frequency (three to seven times). Additional test surveys were conducted at the Parc National de Ranomafana to compare transect methods in an area with known population densities of Eulemur fulvus. Based on these tests, we demonstrate that the use of existing trails for transects can result in a close estimate of local population size, although more replications and longer transects increase precision. Results from regional surveys indicate considerably smaller population densities for E. f. albocollaris (0.086 animals/ha). In contrast, E. f. collaris densities were relatively high (.107 animals/ha) at Midongy‐Sud. We also noted variation among sites in the density of lianas, which was positively correlated with local population density (a possible indication of habitat degradation). More generally, habitats in E. f. albocollaris's range suffered from fragmentation, reduction in forest area, logging, and potentially greater hunting pressure. Based on these results, it is apparent that more immediate steps are necessary to preserve E. f. albocollaris populations and habitats. Am. J. Primatol. 47:51–60, 1999. © 1999 Wiley‐Liss, Inc.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus.

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Mus musculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.