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Abstract Background The lemurs of Madagascar provide an excellent mammalian radiation to explore mechanisms and processes favouring species diversity and evolution. Species diversity, in particular of nocturnal species, increased considerably during the last decade. However, the factors contributing to this high diversity are not well understood. We tested predictions derived from two existing biogeographic models by exploring the genetic and morphological divergence among populations of a widely distributed lemur genus, the sportive lemur (Lepilemur ssp.) along a 560 km long transect from western to northern Madagascar. Results By using the phylogenetic analyses of mtDNA sequence data, molecular diagnostic sites and phenotypic morphometric traits, we uncovered two previously undetected species whose distributions contradict the two existing biogeographic models. Brief species descriptions are provided and a new biogeographic model is proposed (the ”large river model“). Conclusion According to the ”large river model“, large rivers in north and northwestern Madagascar acted as geographical barriers for gene flow and facilitated speciation events on a much smaller spatial scale than previously thought. Thereby, this study does not only show that species diversity in nocturnal Malagasy primates is continuously underestimated but aims to emphasize the need for conservation actions if those species with small ranges shall not face extinction in the near future.

Abstract Background The number of species within the Malagasy genus Lepilemur and their phylogenetic relationships is disputed and controversial. In order to establish their evolutionary relationships, a comparative cytogenetic and molecular study was performed. We sequenced the complete mitochondrial cytochrome b gene (1140 bp) from 68 individuals representing all eight sportive lemur species and most major populations, and compared the results with those obtained from cytogenetic studies derived from 99 specimens. Results Interspecific genetic variation, diagnostic characters and significantly supported phylogenetic relationships were obtained from the mitochondrial sequence data and are in agreement with cytogenetic information. The results confirm the distinctiveness of Lepilemur ankaranensis, L. dorsalis, L. edwardsi, L. leucopus, L. microdon, L. mustelinus, L. ruficaudatus and L. septentrionalis on species level. Additionally, within L. ruficaudatus large genetic differences were observed among different geographic populations. L. dorsalis from Sahamalaza Peninsula and from the Ambanja/Nosy Be region are paraphyletic, with the latter forming a sister group to L. ankaranensis. Conclusion Our results support the classification of the eight major sportive lemur taxa as independent species. Moreover, our data indicate further cryptic speciation events within L. ruficaudatus and L. dorsalis. Based on molecular data we propose to recognize the sportive lemur populations from north of the Tsiribihina River, south of the Betsiboka River, and from the Sahamalaza Peninsula, as distinct species.