Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians).

R-banding chromosomal studies of 21 species of Lemuriformes allowed us to reconstruct the presumed ancestral karyotype of all the Lemuriformes except for Daubentoniidae and permitted the construction of their phylogenetic tree. Chromosome painting with fluorescently labeled heterologous DNA probes permitted comparative chromosome maps to be established. The Zoo-FISH method was used to reassess the karyotypes of 22 species or subspecies. While our results largely confirm the previous reconstruction of the ancestral karyotype, they resulted in a modification of the previously established phylogenetic tree. The Daubentoniidae emerged first followed by the divergence of the families Cheirogaleidae, Indriidae, Lepilemuridae and Lemuridae. Eight chromosome rearrangements occurred in all Lemuriformes except for Daubentoniidae in the common trunk. The present findings do not allow us to propose the occurrence of any rearrangement common to Daubentoniidae and other Lemuriformes, and probably other Prosimii. Conserved syntenies previously described in various mammalian orders were also conserved, while others were specific to the Lemuriformes.

Molecular and cytogenetic evidence for cryptic speciation within a rare endemic Malagasy lemur, the Northern Sportive Lemur (Lepilemur septentrionalis).

Evolutionary relationships of different populations of the threatened malagasy lemur Lepilemur septentrionalis were assessed by sequence analysis of mitochondrial DNA (D-loop region and partial Cyt b gene). One hundred and fifty nine samples were collected from five main different localities in the northern part of Madagascar. We applied the phylogenetic species concept based on fixed diagnostic differences to determine the status of different geographical populations. No nucleotide site diagnoses Ankarana from Andrafiamena or Analamera. However, numerous fixed differences separate Sahafary from all other populations. These results were corroborated by phylogenetic trees. As previous cytogenetic studies, our molecular data suggest that two cryptic species of Lepilemur occur in the extreme north of Madagascar. This speciation is probably caused by chromosomal rearrangements in at least one of the evolutionary lineages. Our study comprises another striking example of how molecular genetic assay can detect phylogenetic discontinuities that are not reflected in traditional morphologically based taxonomies. Our study indicates that the Sahafary population is a hitherto undescribed endangered endemic species which urgently needs conservation efforts.

Taxonomic relationships and sampling effects among Lepilemuridae and Lemuridae using a partial cytochrome b gene.

Partial cytochrome b sequences were used to study relationships between three Lepilemuridae species (Lepilemur dorsalis, L. septentrionalis and L. leucopus) and other Lemuridae species. L. dorsalis were subdivided into two sub-groups, according to their capture area (Nosy-Be island and Sahamalaza peninsula). Relationships deduced from phylogenetic trees as well as genetic distances lead to the classification of the Lepilemurs analysed here into separate species. These Lepilemurs form a monophyletic clade which is the sister clade of all other Lemurs used in this study. Reconstructions using randomly chosen sequences and step by step addition of sequences indicate that phylogenetic results for closely related species need to be analysed with caution, if only a small number of sequences are used to obtain them.

Cytogenetic arguments in favour of a taxonomic revision of Lepilemur septentrionalis.

Cytogenetic investigations performed on 30 specimens of Lepilemur septentrionalis confirmed the existence of 4 karyotypes differing from each other by 1-2 chromosomal rearrangements. These data, pooled with those obtained in earlier studies, showed that out of 60 animals karyotyped only two kinds of hybrids were detected, allowing us to characterise two chromosomally polymorphic populations. No natural hybrids could be found between these two populations, which could thus be considered as two separate species. The possible role of the chromosomal rearrangements in the process of reproductive isolation between these two populations is discussed.

Familial t(6;21)(p21.1;p13) translocation associated with male-only sterility.

A 38-year-old male with primary infertility was referred for cytogenetic investigation. Karyotype analysis revealed a 46,XY,t(6;21)(p21.1;pl3) translocation. The Ag-nucleolar organizer regions (NORs) banding technique demonstrated that the 21p NORs were retained in the derivative and actively transcribed. Family studies showed that three brothers, two sisters and their mother carried the t(6;21). All carrier males suffered from primary infertility with severe oligoasthenoteratospermia or azoospermia, whereas at least two of the three carrier women were fertile. The region of the translocation breakpoint was narrowed down cytogenetically and by fluorescence in situ hybridisation as 21p13 and 6p21.1. Southern blot analysis showed that the gene ZNF165, which maps to this region and which is specifically expressed in the testis, was not disrupted by the translocation. However, studies performed on testicular biopsy showed spermatocyte meiosis anomalies. We discuss the possible mechanisms by which the translocation might affect meiosis in spermatogenesis and lead to infertility.

Lemur chromosomal study with simultaneous R-banding and chromosome painting.

A method for simultaneously obtaining R-banding and chromosome painting is described. It combines fluorescence in situ hybridization with replication of R-bands by 5-bromo-2'-deoxyuridine incorporation into synchronized cells. Distinctive R-banding induced by a modified fluorochrome-photolysis procedure can be observed on both painted and non-painted chromosomes. This method applied to Lemur chromosomes was developed for further studies of chromosomal changes in the evolution of prosimian primates and could also be used in other cytogenetic applications where simultaneous identification of chromosomal R-bands and hybridization signal is needed.

Genetic comparison between different populations of Eulemur macaco flavifrons in northwest Madagascar using RAPD markers.

Eulemur macaco flavifrons, the Sclater's black lemur, is a critically endangered subspecies of northwest Madagascar, which is not yet protected by any reserve. In order to study the feasibility of creating such a reserve, an area of outstanding biological importance was selected in the region of Maromandia-Sahamalaza, which is probably the only remaining place which would permit the long-term survival of the Sclater's black lemur. To determine if genetic management is needed for the Sahamalaza black lemur population, its genetic variability was estimated with random amplified polymorphic DNA (RAPD) markers and compared with other populations. These comparisons demonstrate that the Sahamalaza black lemurs have a genetic variability equivalent to those in other areas. Thus, we conclude that no genetic management is required at the present time.

Transposition of SRY into the ancestral pseudoautosomal region creates a new pseudoautosomal boundary in a progenitor of simian primates.

We have isolated the prosimian lemur homologues for STS and SRY. FISH unambiguously co-localized STS with SHOX, IL3RA, ANT3 and PRK into the meiotic X-Y pairing region (PAR) of lemurs. In contrast to the close proximity of SRY to the pseudoautosomal boundary (PAB) on the Y chromosome in simian primates, SRY maps distant from the PAR in lemurs. Most interestingly, we were able to determine a DNA sequence divergence of 12.5% between the human and lemur SRY HMG box. This divergence directs to a 52 million year period of separate evolution of human and lemur SRY genes. Phylogenetically, this time period falls in between the times that prosimians and New World monkeys branched from the human lineage. Thus, we conclude that approximately 52 million years ago a transposition of SRY into the ancestral eutherian PAR distal to STS and PRK defined a new PAB in a simian progenitor. By this event, STS and PRK, amongst other genes, were excluded from the X-Y crossover process and thus became susceptible to rearrangements and/or deterioration on the Y chromosome in simian primates.

Acoustically dimorphic advertisement calls separate morphologically and genetically homogenous populations of the grey mouse lemur (Microcebus murinus).

Sexual advertisement calls of male mouse lemurs from two neighbouring demes in a dry deciduous forest of western Madagascar were recorded during the breeding season. Demes were located about 1.5 km apart with no geographic barrier between them. They were characterised morphometrically and genotyped by RAPD fingerprinting. According to univariate and multivariate statistical analysis, demes differed neither in body measurements, nor in the banding patterns produced by RAPD fingerprinting. The acoustic pattern of the advertisement call, however, showed significant differences: Six variables of the frequency and time domain differed between the demes. Discriminant function analysis revealed that one variable, total call duration, was sufficient to classify more than 89% of the calls correctly to the corresponding deme. We postulate that these differences are comparable to dialects in birds, because demes were morphologically and genetically indistinguishable and no barrier prevented genetic exchange between them. Possible explanations for the emergence of dialects in a prosimian species are outlined.

Comparison of genetic variability in wild insular and mainland populations of Eulemur macaco: implications for conservation strategy.

DNA fingerprinting analysis was used to investigate genetic variability within and between three wild populations of Eulemur macaco living on two islands and on the mainland. The analyses revealed that the genetic variability of the population from the smaller island was, as expected, lower than that from the larger island. Surprisingly, on the larger island the genetic variability was greater than on the mainland. These results, in agreement with those previously obtained from blood markers, are discussed in relation to the history of these populations. In addition, the study suggests that settlement of the smallest island was not only by animals originating from the mainland, as previously proposed on the basis of blood marker data, but also by lemurs from the nearby, larger, island. Evidence is presented that reliable information about population structure can be derived only from studies using different genetic markers. This information is important to enable appropriate conservation programmes to be designed.

Comparison of the genetic diversity of wild and captive groups of Microcebus murinus using the random amplified polymorphic DNA method.

Continued survival of most animal species depends on population management and active protection. It is generally agreed that, in order to avoid extinction of endangered species, ex situ and in situ conservation must be developed in tandem. However, even though many recommendations have been put forward to promote the survival of captive populations, some rapidly become extinct due to loss of genetic diversity (drift effect). Genetic markers, such as random amplified polymorphic DNA (RAPD) markers, can be applied to rapid testing of many individuals. They also permit analysis of very small amounts of DNA, when small species such as mouse lemurs (Microcebus) are to be tested. Using RAPD markers, we compare genetic diversity in four captive groups of Microcebus murinus to that in a sample of 70 wild mouse lemurs. Following the principles of Mendelian inheritance, each amplified fragment of DNA may be considered as a 'locus' (or an amplifying site). The series of bands amplified by a particular primer in any individual is referred to as the individual's 'profile'. We tested 5 primers, or, in the above terms, we studied 98 different 'loci'. Results showed that the captive groups had lost genetic information with respect to the wild sample. Among the four captive groups, the loss of genetic diversity varied according to their number of founders and/or the management of their captive reproduction. Our study of polymorphism permitted us to establish tools for the genetic management of captive breeding, and for the determination of paternity which frequently give better results than behavioural studies; and simulation of introductions or departures of individuals in one very monomorphic group permitted estimation of future increases in its genetic diversity.

Y-autosome translocation and infertility: usefulness of molecular, cytogenetic and meiotic studies.

An apparently balanced reciprocal translocation 46,X,t(Y;6) (q11.23 approximately q12;p11.1) was observed in an infertile man with severe oligozooteratozoospermia. Different mitotic chromosome banding patterns were performed and fluorescence in situ hybridization indicated a breakpoint in the fluorescent Yq heterochromatin. Molecular genetic deletion experiments for the azoospermia factor region in distal Yq11 showed the retention of the DAZ gene and meiotic pairing configurations suggested that the man's infertility could be due to the pairing behaviour of the Y;6 translocation chromosome with the X chromosome visualised by synaptonemal complex analysis at the electron microscopy level. The morphological appearance of the normal chromosome 6 and the Y;6 translocated chromosome included in the compartment of the sex vesicle may allow an explanation of the degeneration of most spermatocytes after the pachytene stage.

Phylogenetic relationships among Indriidae (Primates, Strepsirhini) inferred from highly repeated DNA band patterns.

Comparative studies of highly repeated DNA from different species of Indriidae (Primates, Strepsirhini) allowed confirmation of the specific status of Avahi occidentalis, A. laniger and Propithecus tattersalli. The comparison of their band patterns revealed the existence of specific and common bands from which a cladogram of the family is inferred. This cladogram shows that Avahi clade is the sister-group of Indri and Propithecus clade, and that P. verreauxi is related to P. diadema. These results were discussed in view of those obtained from cytogenetic, morphological and molecular data (mitochondrial DNA). This study shows the capacity of the repeated sequence pattern comparison to be used as a tool for confirming taxa status, (taxinomic classification is a primary determinant of management priorities for endangered species, neglect of distinct taxa may lead to their extinction), and for inferring phylogenetic relationships among related species.

Meiotic study of hybrids in the genus Eulemur and taxonomic considerations.

A reproductive study was conducted on seven hybrids of Eulemur showing chromosomal multivalents involving at least four chromosomes at the pachytene stage. Three individuals were infertile hybrids and one presented a reduced spermatogenesis. In three out of these four hybrids, multivalents were associated with the sex bivalent in a large number of spermatocytes (23%). The relative importance of the reduction of fertility in males linked to chromosomal multivalent formation as well as the genetic background is discussed with regard to the use of cytogenetic data for systematics. Our findings argue for the classification of Eulemur fulvus collaris and E. f. albocollaris in two separate species. In regard to their repartition area, their separation along a linear north-south axis in Madagascar is discussed.

Chromosome painting: a method for testing chromosomal changes in lemur evolution.

Chromosome painting using commercially available human chromosome-specific DNA libraries was performed to elucidate chromosomal rearrangements in lemur evolution. Human-specific probes for chromosomes 3, 14, 15, and 21 were used to paint chromosomes of six species: Eulemur fulvus mayottensis, Varecia variegata, Lemur catta, Hapalemur simus, H. griseus griseus, and H. aureus. All human chromosome libraries hybridized specifically to chromosome segments of varying length or to whole arms of Lemur chromosomes. The labeling was clearly visualized and permitted precise delineation of the hybridized Lemur chromosomal segments. The use of commercial probes of human chromosomes for chromosome painting appears efficient enough to investigate homology in different species of Lemur. In general, the results obtained by chromosome painting in this study confirm results previously obtained by the R-banding technique but modify the location of some chromosomal rearrangements on different branches of the evolutionary tree of the Lemuridae and reveal some new rearrangements that were not detectable with banding techniques. These results show that chromosome painting with human chromosome-specific DNA libraries can provide useful information in comparative studies on karyotypes of distantly related mammalian species, providing a powerful tool for evolutionary studies, especially in phylogeny.

Non-telomeric chromosome localization of (TTAGGG)n repeats in the genus Eulemur.

The chromosomal distribution of the (TTAGGG)n telomeric repetitive sequences was studied in the Malagasy species Eulemur fulvus fulvus (2n = 60), Eulemur rubriventer (2n = 50), Eulemur coronatus (2n = 46) and Eulemur macaco (2n = 44). These sequences hybridize to the telomeres of all chromosomes of the four species and also to the pericentromeres of all chromosomes of E. fulvus, E. coronatus and E. macaco, with the exception of the pericentromeres of E. coronatus and E. macaco chromosomes 9, the homeologous E. fulvus chromosomes 2 and E. macaco chromosomes 1. In E. rubriventer only a very weak signal was detected at the pericentromeres of a few chromosomes. In E. fulvus, E. coronatus and E. macaco, non-telomeric (TTAGGG)n sequences collocalize with constitutive heterochromatin. The interspecific differences of the hybridization pattern of (TTAGGG)n sequences at the pericentromeres suggest that E. rubriventer branched off the common trunk before amplification of endogenous (TTAGGG)n sequences occurred in pericentromeric regions.