During the last decade, comparisons of mitochondrial DNA sequences have been very useful for the analyses of the phylogenetic species relationships including lemurs [11
]. In our study, cytochrome b and 12S mtDNA sequences were used, in order to clarify the position of each species and subspecies in the genus Hapalemur.
Despite the fact that we analysed only short sequences, the genetic distances are considered to be relevant because of the large number of animals involved, since large sample size reduces errors in the estimation of evolutionary relatedness [11
Comparisons of cytochrome b sequences demonstrated that pairwise genetic distances between H. simus and H. aureus and the other Hapalemur (distances ranging from 0.09098 to 0.15338) are the highest. The corresponding phylogram also shows that these taxa are well separated. Our molecular data thus strongly support the species status for H. simus, H. aureus and H. griseus.
Among the H. griseus,
the genetic divergence observed between H. g. meridionalis
and the HGG, HGA, HGsspa
and HGO (ranging from 0.06240 to 0.10473) is markedly higher than all other values of the same level of intra H. griseus
comparison (ranging from 0.00281 to 0.03781). The phylogram also clearly separates the H. g. meridionalis
from other H. griseus.
This high genetic divergence in the ranging of those observed between species of lemurs, would support the classification of H. g. meridionalis
in a separate species "H. meridionalis"
despite the relatively small chromosomal differences observed [9
Lower distances (ranging from 0.01133 to 0.03781) are observed between H. griseus
and H. g. occidentalis.
Moreover, these two forms cluster in the same clade. So, despite the cytogenetic differences existing between these two forms, which differ by two chromosomal rearrangements [10
], we propose the maintenance of the subspecies status for H. g. occidentalis.
On the basis of our analyses, H. g. alaotrensis
is indistinguishable from H. g. griseus.
The very short genetic distances found between H. g. griseus
and H. g. alaotrensis
(0.00281 to 0.00847) suggests a combination of these two groups into a single subspecies. However, the differences of the body sizes [4
] and the differences in the content of heterochromatin found in both karyotypes [22
] support the separation of H. g. griseus
and H. g. alaotrensis
in separate subspecies [23
], but in no case in species apart as it has been previously proposed [24
The systematic position of HGssp is more difficult to establish when cytogenetic and molecular data are compared. Cytogenetic data demonstrated the existence of an unique chromosomal polymorphic subspecies characterized by two karyotypes, 2N = 56 and 2N = 54, and their hybrids 2N = 55 [8
]. In the view of molecular data, HGssp contains two groups, HGsspa
, separated by an important genetic distance (0.06240–0.09719) in the range of those observed between species. Each of these two groups contains both karyotypes, 2N = 56 and 2N = 54, as well as their hybrids. The HGsspa
are separated from HGG, HGA and HGO by a genetic distance in the range of those observed between subspecies (0.00563–0.03781), while HGsspb
appears similar to HGM. Moreover, the HGsspb
haplotypes are mixed with those of HGM. Taking into account these results, HGsspa
should be considered as belonging to the group of HGG, HGA and HGO, and HGsspb
as belonging to the group of HGM.
The existence of two well-separated clades among the Hapalemur griseus ssp
originating from the Ranomafana region could possibly resulted from either recent mitochondrial DNA introgression or ancestral polymorphism. As the boundaries between HGM and HGssp are still unknown, we could hypothesize that the limits could be close to Ranomafana, allowing hybridization between these two forms. A transfer of mitochrondrial DNA from HGM into the HGssp population could have occurred through a matrilinear process resulting in the HGsspb
haplotype. New investigations in areas located in the south of Ranomafana should thus allow the finding of HGM populations containing introgressed HGssp mitochondrial DNAs, unless this transfer occurs only from HGM to HGssp. A second hypothesis is that an ancestral polymorphic population containing both HGM and HGssp haplotypes and a chromosomal polymorphism have diverged in two separated populations. In the population of Ranomafana, the HGM and HGssp haplotypes as well as the chromosomal polymorphism were maintained. In the second population, only the HGM haplotype remained present, and a gain of a large block of heterochromatin gave rise to the karyotype characteristic of the HGM [9
The comparisons of the phylogenetic trees based on mitochondrial DNA sequences with those previously obtained from cytogenetic data are only partially concordant. In both trees, H. simus
emerges first, followed by H. aureus
and then by the different H. griseus
forms. The cytogenetic data allow to propose an evolutionary tree in which H. g. occidentalis
emerges first, followed by H. g. griseus,
and H. g. meridionalis
], whereas on the tree based on cytochrome b and 12S sequences, H. g. meridionalis
appears as a sister clade of the other H. griseus.
This difference may be related to the short distances observed inside each clade which allowed no branching order.