Genotyping-by-sequencing (GBS) and related methods are increasingly used for studies of non-model organisms from population genetic to phylogenetic scales. We present GIbPSs, a new genotyping toolkit for the analysis of data from various protocols such as RAD, double-digest RAD, GBS, and two-enzyme GBS without a reference genome. GIbPSs can handle paired-end GBS data and is able to assign reads from both strands of a restriction fragment to the same locus. GIbPSs is most suitable for population genetic and phylogeographic analyses. It avoids genotyping errors due to indel variation by identifying and discarding affected loci. GIbPSs creates a genotype database that offers rich functionality for data filtering and export in numerous formats. We performed comparative analyses of simulated and real GBS data with GIbPSs and another program, pyRAD. This program accounts for indel variation by aligning homologous sequences. GIbPSs performed better than pyRAD in several aspects. It required much less computation time and displayed higher genotyping accuracy. GIbPSs retained smaller numbers of loci overall in analyses of real GBS data. It nevertheless delivered more complete genotype matrices with greater locus overlap between individuals and greater numbers of loci sampled in all individuals.

Unequal degrees of taxonomic subdivision can pose problems for research that relies on cross-taxon comparisons of biogeographic patterns. Numerous species of lemurs have been described in recent years. These descriptions were unevenly distributed over the genera of lemurs as exemplified by the closely related mouse lemurs (Microcebus spp.) and dwarf lemurs (Cheirogaleus spp.). According to previous studies, these genera display striking differences such as many versus few species, small versus large distributions, and small versus large mitochondrial divergence within and between species. We questioned if these differences reflect the biological reality or a biased taxonomic subdivision, which might be partially due to relatively small amounts of available genetic data from dwarf lemurs. We complemented existing datasets with genetic data from 51 dwarf lemurs from nine sites in southern Madagascar. We analyzed the mitochondrial cytb gene and the nuclear loci adora3, fiba, and vWF. Based on a comparison of mitochondrial genetic data from both genera, we delineated eight hypothetical subgroups within two recognized Cheirogaleus species. We used mitochondrial and nuclear data to reconstruct species trees and to estimate divergence times between Microcebus species and Cheirogaleus subgroups. We further performed Bayesian species delimitations based on nuclear sequence data from Cheirogaleus subgroups. Strong signals in mitochondrial and nuclear data indicate the existence of deeply divergent, distinct groups within recognized Cheirogaleus species. Based on several lines of evidence, we conclude that the species diversity in Cheirogaleus has been underestimated so far. We delineate six species among the eight subgroups and provide a formal description for one new Cheirogaleus species.

BACKGROUND:Several mechanistic models aim to explain the diversification of the multitude of endemic species on Madagascar. The island's biogeographic history probably offered numerous opportunities for secondary contact and subsequent hybridization. Existing diversification models do not consider a possible role of these processes. One key question for a better understanding of their potential importance is how they are influenced by different environmental settings. Here, we characterized a contact zone between two species of mouse lemurs, Microcebus griseorufus and M. murinus, in dry spiny bush and mesic gallery forest that border each other sharply without intermediate habitats between them. We performed population genetic analyses based on mtDNA sequences and nine nuclear microsatellites and compared the results to a known hybrid zone of the same species in a nearby wide gradient from dry spiny bush over transitional forest to humid littoral forest.RESULTS:In the spiny-gallery system, Microcebus griseorufus is restricted to the spiny bush; Microcebus murinus occurs in gallery forest and locally invades the dryer habitat of its congener. We found evidence for bidirectional introgressive hybridization, which is closely linked to increased spatial overlap within the spiny bush. Within 159 individuals, we observed 18 hybrids with mitochondrial haplotypes of both species. Analyses of simulated microsatellite data indicate that we identified hybrids with great accuracy and that we probably underestimated their true number. We discuss short-term climatic fluctuations as potential trigger for the dynamic of invasion and subsequent hybridization. In the gradient hybrid zone in turn, long-term aridification could have favored unidirectional nuclear introgression from Microcebus griseorufus into M. murinus in transitional forest.CONCLUSIONS:Madagascar's southeastern transitional zone harbors two very different hybrid zones of mouse lemurs in different environmental settings. This sheds light on the multitude of opportunities for the formation of hybrid zones and indicates an important influence of environmental factors on secondary contact and hybridization. Our findings suggest that hybridization could enhance the adaptability of mouse lemurs without necessarily leading to a loss of distinctiveness. They point to a potential role of hybridization in Madagascar's diversification history that requires further investigation.

The lemurs of Madagascar are known for their extraordinary levels of speciation. However, the mechanisms and environmental conditions that led to this diversity remain obscure. We used 3 species of Microcebus (M. griseorufus, M. murinus, M. rufus) occurring along an environmental gradient as a model to investigate 1) how the different species are distributed in relation to variation in environmental conditions and ecotones; 2) whether or not the morphology of a given species varies in relation to environmental conditions; and 3) whether or not there is evidence for morphological character displacement to reduce congeneric competition in sympatry vs. allopatry. The 3 species of Microcebus show clear associations with specific habitat types. Distributions overlap at ecotones. Nevertheless, the ecotone between dry spiny and gallery forest represents a species boundary between Microcebus griseorufus and M. murinus while the ecotone between dry spiny forest and evergreen humid forest represents the species boundary between M. murinus and M. rufus. Different ambient conditions are not reflected in changes in body measurements of Microcebus murinus living in different vegetation formations. There is no indication for character displacement in sympatry vs. allopatry. Thus, differences in body mass or other morphological characteristics do not contribute to species separation between Microcebus griseorufus and M. murinus. The results confirm the importance of ecotones as species boundaries as a condition postulated for the radiation of lemur and other species on Madagascar. They also demonstrate different habitat affinities of seemingly very similar lemur species and thus illustrate our very limited understanding of the actual selection pressures, adaptations of lemurs to their environments, and their possible response to interspecific competition.

Hybrid zones in ecotones can be useful model systems for the study of evolutionary processes that shape the distribution and discreteness of species. Such studies could be important for an improved understanding of the complex biogeography of Madagascar, which is renowned for its outstanding degree of small-scale endemism. Certain forest remnants in central Madagascar indicate that transitional corridors across the island could have connected microendemics in different forest types in the past. Evolutionary processes in such corridors are difficult to study because most of these corridors have disappeared due to deforestation in central Madagascar. We studied a hybrid zone in one of the few remaining ecotonal corridors between dry and humid forests in Madagascar, which connects two species of mouse lemurs, Microcebus griseorufus in dry spiny forest and Microcebus murinus in humid littoral forest. We sampled 162 mouse lemurs at nine sites across this boundary. Morphometric analyses revealed intermediate morphotypes of many individuals in transitional habitat. Bayesian clustering of microsatellite genotypes and assignment tests yielded evidence for a mixed ancestry of mouse lemurs in the ecotone, where we also observed significant linkage disequilibria and heterozygote deficiency. In contrast to these observations, mitochondrial haplotypes displayed a sharply delimited boundary at the eastern edge of spiny forest, which was noncoincident with the signals from microsatellite data. Among several alternative scenarios, we propose asymmetric nuclear introgression due to male-biased dispersal, divergent environmental selection, and an expansion of dry spiny forest in the course of aridification as a probable explanation of our observations.

A basic understanding of the taxonomy, diversity, and distributions of primates is essential for their conservation. This review of the status of the taxonomy of lemurs is based on a 5-d workshop entitled “Primate Taxonomy for the New Millennium,” held at the Disney Institute, Orlando, Florida, in February 2000. The aim is not to present a taxonomic revision, but to review our current understanding of the diversity and current and past ranges of lemurs and indicate where there is controversy, discrepancy, or lack of knowledge. Our goal therefore is to provide a baseline for future taxonomic investigation, as well as a clearer focus for research and conservation priorities. We here focus on the lemurs of Madagascar and recognize 5 families, 15 genera, and 99 species and subspecies. We list 39 species of lemurs described since 2000: 2 dwarf lemurs, Cheirogaleus; 11 mouse lemurs, Microcebus; a giant mouse lemur, Mirza; a bamboo lemur, Hapalemur; 17 sportive lemurs, Lepilemur; and 7 woolly lemurs, Avahi. Taxonomic revisions have resulted in the resurrection of a further 9 taxa. However, the figures do not represent the total diversity of Malagasy lemurs because more new species are being identified via new field studies and accompanying genetic research, and should be described in the near future.

Seven species of dwarf lemurs (Cheirogaleus spp.) are currently recognized after a recent revision of the genus. During a field study in southeastern Madagascar, we observed 3 distinct morphotypes of Cheirogaleus resembling Cheirogaleus medius, C. major and C. crossleyi. In particular, for Cheirogaleus crossleyi southeastern Madagascar was far away from the known distribution range of the species when referring to the recent revision of the genus. In order to clarify the taxonomic status of the 3 morphotypes, we compared field samples from southeastern Madagascar to museum specimens from several locations in Madagascar and to field samples from Kirindy/CFPF, western Madagascar by genetic analyses using the mitochondrial cytochrome b gene. Genetic data yield strong support for 2 of the 3 morphotypes to represent Cheirogaleus medius and C. major. The third morphotype is genetically closely related to Cheirogaleus crossleyi, albeit more distantly than the other 2 morphotypes to the respective reference samples. The genetic distance is related to a considerable geographic distance between the location of our field study and the origin of the respective reference specimen, because Cheirogaleus crossleyi had been reported only from northern Madagascar. Our field observations indicate an isolated population in southeastern Madagascar. We propose to identify them as Cheirogaleus crossleyi and discuss the population genetic and biogeographic considerations supporting this conclusion.

We report the development of three new microsatellites and four transferred across-species for fork-marked lemurs (Phaner furcifer). Two markers were isolated from Cheirogaleus medius and one from Microcebus murinus. The transferred markers also originate from Cheirogaleus medius and Microcebus murinus. The seven markers were tested on 30 individuals of Phaner furcifer and have proven to be useful for inclusion and exclusion of potential parents. The markers presented here are the first published for application on, Phaner furcifer.

We report the development of 13 new microsatellite markers for mouse lemurs (Microcebus sp.). Two markers were isolated from the fat tailed dwarf lemur (Cheirogaleus medius) and 11 from the grey mouse lemur (Microcebus murinus). A total of 561 individuals from four different species of mouse lemurs was genotyped with the newly developed markers. All markers showed Mendelian inheritance in 21 families of mouse lemurs. All markers show polymorphism in several species of mouse lemurs and seven amplified in C. medius. Among these new markers are the first 10 published for M. berthae and the first 11 for M. griseorufus.

The hamadryas baboon, Papio hamadryas hamadryas, represents a rare exception from the pattern of female philopatry and male-biased dispersal predominant in mammals including primates. To elucidate the possible consequences of the dispersal pattern on the population genetic structure of hamadryas baboons, we sequenced the maternally transmitted mitochondrial hypervariable region I of 74 individuals from ten sampling locations in different ecogeographic zones of Eritrea. To this end, individual fecal samples were collected at sleeping cliffs. Upon comparing the individual sequences by means of phylogenetic tree reconstructions and AMOVA, we could not detect a population genetic structure corresponding to a geographic pattern. Tree reconstructions revealed the existence of two profoundly different lineages both present at most of the sampling locations. These findings and Mantel correlations of genetic distances and the frequency of shared haplotypes to geographic distances point to the presence of female dispersal. Female-mediated gene flow is detectable over geographic distances exceeding those between neighboring subpopulations. Our study therefore corroborates local behavioral observations on a broad geographic scale. After inclusion of geographically closely situated olive baboons, P. h. anubis, in the analyses, all anubis sequences fell within one hamadryas clade. Possible scenarios leading to this situation including long-term hybridization processes are discussed.