The genus Hapalemur of the Malagasy Prosimian family Lemuridae comprises three clearly different species: Hapalemur griseus1, H. simus2 and H. aureus3. Classification of different types within the H. griseus group has been controversial, particularly in the light of the variations existing in coat colour 4. Systematic cytogenetic studies led to the description of four subspecies of H. griseus characterised by different karyotypes: H. g. griseus (HGG) (2N = 54) 56, initially considered as the most widespread and supposed to live in the Eastern forest of Madagascar from the north to the south 7; H. g. occidentalis (HGO) (2N = 58) 6 restricted in the north-west and the eastern part of Madagascar as east as Maroantsetra 48; H. g. meridionalis (HGM) (2N = 54) 9 found in the south, near Fort Dauphin, and differing from H. g. griseus in one metacentric chromosome ; H. g. alaotrensis (HGA) (2N = 54) 6, restricted to the bed of the Alaotra lake, with a karyotype similar to HGG containing more juxtacentromeric heterochromatin, and characterized by a larger body size 4. In addition, a new variant of HG, H. griseus ssp (HGssp), has recently been described. This type is characterized by a chromosomal polymorphism (2N = 54, 55 and 56) and is living in the area of Ranomafana-Kianjavato 8.

The comparison of the karyotypes of the different Hapalemur led to a phylogenetic tree characterised by an earlier emergence of H. simus (HSI), followed by H. aureus (HAU) which represents the sister clade of the different groups of the H. griseus subspecies 810. As molecular studies were fruitfully used for taxonomic investigations of several taxa, including lemurs 1112131415, and as no large morphological differences could be found between the cytogenetically close subspecies of H. griseus, mitochondrial cytochrome b and 12S DNA analyses were performed in order to determinate more accurately their species and/or subspecies status.

A total of 115 animals covering all the genus Hapalemur were captured in bamboo forests extending from the north to the south of Madagascar. The different capture areas of each species and subspecies as the number of animals are indicated in Fig. 1.

The PCR amplification of the cytochrome b resulted in a 357 bp fragment. Alignments using blast search demonstrated that this fragment corresponds to the beginning of the mitochondrial cytochrome b gene. All the sequences were aligned and the substitutions were scored. Animals sharing the same sequences were regrouped into a same haplotype. These different haplotypes are summarised in Fig. 1 and table 2. Consensus sequences constructed with the most common sequence in each species and subspecies (not shown) demonstrated that transition substitutions occur more frequently than transversions (an average of eight transitions for 1 transversion) and gaps are absent. As expected 16, most of the substitutions occur on the third position of the codon (54 out of 64 substitutions), while the first position changed nine times and the second once. The portion amplified comprises the four invariant codons considered necessary for cytochrome b function 17. Changes were sometimes noticed in the third base of the codon in these positions, but the amino acid remains the same so that the functionality of the cytochrome b is not affected.

Kimura two parameter distances 18 and absolute distances calculated with the different haplotypes (Table 1) as phylogenetic trees constructed with the computer program Pylogenetic Analysis Using Parsimony (PAUP) 4.0b.4a 19 (Figs 2, 3, 4, 5) were used to compare the different Hapalemur species and subspecies. Neighbor-joining analyses of cytochrome b haplotypes demonstrated that H. simus emerged first followed by H. aureus which is the sister clade of the H. griseus subspecies. Inside H. griseus subspecies, two subclades are present and the H. griseus ssp (HGssp) are distributed in both the subclades (Figs 2, 3). For this reason, the H. griseus ssp were separated in HGssp_a and Hgssp_b. The first subclade (S1) contains HGA, HGG, HGssp_a and HGO. The second subclade (S2) is composed of HGM and HGssp_b. Maximum parsimony analyses (data not shown) demonstrated the same topology, unless the bootstrap support for S1 dropped from 94 to 66.

The use of the Kimura two parameter method (Table 1) shows inside S1 a very close distance between HGA and HGG (ranging from 0.00281 to 0.00847), while a larger distance occurs between HGG and HGssp_a (0.00847 to 0.02875) as well as between HGG and HGO (0.01412 to 0.03180). In S2 (Table 1), short distances are observed between HGM and HGssp_b (0–0.02855), (in this subclade, the sequence of HGM 02 and HGssp 01 are identical). Larger distances ranging from 0.10473 (HGssp_a/HGM) to 0.06240 (HGM-HGssp_b/HGG-HGssp_a) separates S1 from S2 (Table 1).

In order to confirm the position of H. griseus ssp inside the H. griseus subclades, a portion of 12S mitochondrial DNA was amplified and sequenced. The resulted 438 bp fragments were aligned, and the sequences were analyzed with the program PAUP. The trees obtained with HGO, HGG, HGssp and HGM 12S sequences (Figs 4, 5) also demonstrated a separation of HGssp into two subclades. The first (HGssp_a), which is composed of animals clustering with HGO and HGG when cytochrome b sequences are analyzed (Figs 2, 3), also clusters with HGO and HGG when 12S sequences are employed (Figs 4, 5). The second (HGssp_b), constructed with animals clustering with HGM when cytochrome b sequences are analyzed (Figs 2, 3), also clusters with HGM when 12S sequences are employed (Figs 4, 5).

During the last decade, comparisons of mitochondrial DNA sequences have been very useful for the analyses of the phylogenetic species relationships including lemurs 111213141520. 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 1121.

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, HGssp_a 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, HGssp_a and HGssp_b, 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 HGssp_a are separated from HGG, HGA and HGO by a genetic distance in the range of those observed between subspecies (0.00563–0.03781), while HGssp_b appears similar to HGM. Moreover, the HGssp_b haplotypes are mixed with those of HGM. Taking into account these results, HGssp_a should be considered as belonging to the group of HGG, HGA and HGO, and HGssp_b 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 HGssp_b 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. meridionalis10, 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.

Our molecular studies of the Hapalemur genus raise the question of the classification of H. g. meridionalis in the species status H. meridionalis. They also confirm the subspecies status of H. g. occidentalis and the absence of arguments in favour of the classification of H. g. alaotrensis as a separate species. The sequencing of the Hapalemur griseus ssp originating from Ranomafana reveals animals clustering either with the H. meridionalis or with the group of Hapalemur griseus griseus. No monophyletic clade could be determined in this new cytotype, so that the taxonomic status of the Hapalemur griseus ssp remains undefined. As our molecular data did not match the branching sequence within the H. griseus group based on cytogenetic data, further investigations including nuclear DNA will be necessary in order to resolve this issue.