Introduction

This study investigates the systematics of the lemurs of Madagascar. Five lemur families are currently recognised, but their taxonomy, nodal relationships and composition need clarification. The families Lepilemuridae and Daubentoniidae each contain only 1 genus (Lepilemur and Daubentonia). The family Indridae is classified into 3 genera (Avahi, Indri and Propithecus). Propithecus includes 3 species containing up to 10 described subspecies, whose evolutionary relationships remain contentious. In particular, it is unclear whether P. verreauxi deckeni and P.v. coronatus populations are differentiated at the subspecific level. Furthermore, the taxonomic status of the recently discovered P. tattersalli also requires further examination. The family Cheirogaleidae is currently classified into 8 species in 5 genera, whose phylogenetic relationships have yet to be clarified. Taxonomic status of Mirza coquereli, Allocebus trichotis and the recently discovered Microcebus ravelobensis require further examination. The family Lemuridae includes 4 genera. The taxonomy and phylogenetic relationships between Lemur, Eulemur and Hapalemur, and of Varecia to the other lemurids continue to be hotly debated. Nodal relationships among the 5 Eulemur species also remain uncertain. The phylogenetic relationships among Hapalemur species and subspecies as well as their taxonomic status need to be verified. Eulemur fulvus includes 7 subspecies, whose evolutionary relationships remain a matter for debate. 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.

Methods

A mitochondrial DNA sequence data set from the ND3, ND4L, ND4 genes and 5 tRNAs (Gly, Arg, His, Ser, Leu) was generated to try to clarify phylogenetic relationships among lemur families, genera, species and subspecies. To attempt this goal, a total of 131 lemurs from 12 genera, 25 species and 18 subspecies have been sequenced. Two galagos were included as outgroup taxa. The ~2400 bp sequences were analysed using maximum parsimony, neighbor-joining and maximum likelihood methods. Different weighting schemes and outgroups have been applied in trying to resolve phylogenetic relationships.

Results and Discussion

The mitochondrial DNA sequence data used in this study yield a strong phylogenetic signal. A number of clear findings emerged: The data strongly support the monophyly of the Malagasy lemurs. Regardless of which outgroup was applied or how the data set was weighted, Daubentonia consistently groups as sister to a clade containing the other 4 lemur families. However, the molecular data failed to yield clear resolution of phylogenetic relationships among the 4 families Cheirogaleidae, Indridae, Lemuridae and Lepilemuridae. Nevertheless, the monophyly of each of the 5 lemur families is supported in all analyses.

Lepilemur is indeed deeply separated from all other lemur genera in the data set, which strongly supports the family status (Lepilemuridae) of this single genus. Tree topology and pairwise genetic distances clearly confirm specific status for L. edwardsi, L. ruficaudatus and L. septentrionalis. Paraphyly and a high degree of genetic divergence among L. edwardsi individuals clearly suggests that two species exist in the range of the currently recognised species L. edwardsi.

In Cheirogaleidae, Mirza and Microcebus form a clade representing the sister group of Allocebus, while a clade containing Cheirogaleus major and C. medius diverges first. M. ravelobensis and M. rufus form a subclade within Microcebus, with M. murinus as its sister group. Pairwise distance comparisons and tree topology support the generic status of Mirza coquereli and species-level divergence of M. ravelobensis. Furthermore, 'M. rufus' may well represent more than one species.

In Indridae, all analyses group Avahi as sister to the clade containing all Propithecus. P. diadema is the first species to diverge within the genus Propithecus. Among the remaining Propithecus, one subclade is formed by P.v. coquereli and P. tattersalli, while P.v. verreauxi, P.v. deckeni and P.v. coronatus form the second subclade. All analyses fail to resolve P.v. coronatus and P.v. deckeni into separate monophyletic lineages. Based on pairwise distance comparisons and tree topology, it is concluded that P. tattersalli does not represent a distinct species and that P.v. deckeni and P.v. coronatus do not deserve subspecific rank. On the other hand, these analyses indicate that P.v. coquereli may well represent a distinct species.

In Lemuridae, the results support monophyly of Eulemur, a basal divergence of Varecia, and a sister-group relationship for Lemur/Hapalemur. Based on tree topology and pairwise distance comparisons, it is concluded that Varecia and Eulemur both represent distinct genera separate from L. catta. H. griseus and H. aureus form a clade, but the sequence data do not permit 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 resolve clearly relationships among those 3 species or with the more distantly related E. coronatus and E. macaco. The 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 high genetic distances among individual V.v. variegata indicate that there may be more phylogenetic structure within this taxon than is indicated by current taxonomy. The sequence data do not yield clear resolution of H.g. griseus from H.g. alaotrensis. Considerable genetic differentiation exists among the small sample of H.g. occidentalis individuals examined here, indicating that more than one subspecies may exist along the western coast.

Analyses resolved 34 E. fulvus specimens into 6 clades: ((albocollaris, collaris) (rufus (rufus (fulvus/mayottensis (albifrons/fulvus/sanfordi))))). It can be concluded that E.f. albocollaris and E.f. collaris do not represent distinct species 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.

Literature

Pastorini J (2000) Molecular Systematics of Lemurs. Ph.D. thesis, University of Zürich.

Pastorini J, Forstner MRJ & Martin RD (2000) Relationships among brown lemurs (Eulemur fulvus) based on mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 16:418-429. Abstract/Download

Pastorini J, Forstner MRJ & Martin RD (2001) Phylogenetic history of sifakas (Propithecus: Lemuriformes) derived from mtDNA sequences. American Journal of Primatology 53:1-17. Abstract/Download

Pastorini J, Martin RD, Ehresmann P, Zimmermann E & Forstner MRJ (2001) Molecular phylogeny of the lemur family Cheirogaleidae (Primates) based on mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 19:45-56. Abstract/Download

Pastorini J, Forstner MRJ & Martin RD (2002) Phylogenetic relationships among Lemuridae (Primates): evidence from mtDNA. Journal of Human Evolution 43:463-478. Abstract/Download

Pastorini J, Forstner MRJ & Martin RD (2002) Phylogenetic relationships of the gentle lemurs (Hapalemur). Evolutionary Anthropology 11(S1):150-154. Abstract/Download

Pastorini J, Thalmann U & Martin RD (2003) A molecular approach to comparative phylogeography of extant Malagasy lemurs. Proceedings of the National Academy of Science of the USA 100:5879-5884. Abstract/Download

Collaborators

Prof. Robert D. Martin
The Field Museum, Chicago, IL, USA

Dr. Urs Thalmann
Anthropological Institute, University of Zürich, Zürich, Switzerland

Prof. Michael R.J. Forstner
Texas State University, San Marcos, TX, USA

Lepilemur edwardsi

Propithecus verreauxi coronatus

Eulemur coronatus

Varecia variegata rubra

Eulemur fulvus rufus